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Annu. Rev. Biochem.

Annu. Rev. Biochem. 1995. 64:403-34 Copyright Q 1995 by Annual Reviews Inc. All righls reserved

COLLAGENS: Molecular Biology, Diseases, and Potentials for Therapy

Darwin J. Prockop

Department of Biochemistry and Molecular Biology, Jefferson Institute of

Molecular Medicine, Jefferson Medical College of Thomas Jefferson University,

Philadelphia, Pennsylvania 19107

Kari I. Kivirikko

Collagen Research Unit, Biocenter and Department of Medical Biochemistry,

University of Oulu, 90220 Oulu, Finland

KEY WORDS:

fibrosis, osteogenesis imperfecta, transgenic mice, gene therapy, antisense

oligonucleotides, antisense gene

CONTENTS

THE COLLAGEN FAMILY OF PROTEINS AND GENES

 

404

the Collagen

Triple Helix

 

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404

Siructure and Functions of Types of Collagen

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BIOSyNTHESIS

General Features

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405

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Intracellular Processing

 
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Extracellular Events ••

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Potentials for Inhibiting Fibrosis

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MUTATIONS IN MEN AND MICE

 

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Mutations in Patients

 

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MUiations in Transgenic

 

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Potentials for Gene Therapy

 

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A BSTRACT

The collagen superfamily of proteins now contains at least 19 proteins formally defined as collagens and an additional ten proteins that have collagen-like domains. The most abundant collagens form extracellular fibrils or network­

like structures, but the others fulfill a variety of biological functions. Some of

403

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Annu. Rev. Biochem.

404 PROCKOP & KlVIRlKKO

the eight highly specific post-translational enzymes involved in collagen bio­

synthesis have recently been cloned. Over 400 mutations in 6 different colla­

gens cause a variety of human diseases that include osteogenesis imperfecta,

chondrodysplasias, some fonns of osteoporosis, some fonns of osteoarthritis,

and the renal disease known as the Alport syndrome. Many of the disease

phenotypes have been produced

genes. There has been increasing interest in the possibility that the unique

post-translational enzymes involved in collagen biosynthesis offer attractive targets for specifically inhibiting excessive fibrotic reactions in a number of diseases. A number of experiments suggest it may be possible to inhibit

collagen synthesis with oligo- nucleotides or antisense genes.

in transgenic mice with mutated collagen

THE COLLAGEN FAMILY OF PROTEINS AND GENES

At least 19 proteins are now known as collagens, and at least an additional 10 proteins have collagen-like domains. Initially, collagens were defined as pro­ teins of the extrncellular matrix that contained large domains comprised of repeating -Gly-X-Y- sequences and that folded into a unique triple-helical structure. Screening of cDNA and genomic DNA libmrles with probes for

collagens revealed a large number of related proteins with varying lengths of

repeating

collagens, the proteins encoded were defined as collagens, even though in

-Gly-X- Y- sequences. Because they were discovered in searches for

some cases the triple-helical domains were small and most of the protein

structure was globular. Also, a few proteins studied in other contexts were found to contain triple-helical domains but were not defined as collagens. The

variety in the structures of different collagens and related proteins implies that

they have vastly different biological functions. Because of the extensive literature on collagens, in this chapter we concen­

tmte primarily on recent advances in the field. For more detailed data, the reader is referred to severnl previous reviews on the structures and functions of collagens (1-11), on the structures of collagen genes (12-14), on the bio­ synthesis of the proteins (1, 6, 7, 15, 16), and on mutations in patients and

mice (2, 17-26). A vast liternture is also available on cis-regulatory elements for collagen genes, and several tmnscription factors have been characterized. The topic, however, is not reviewed here, in part because there are conflicting

data from similar experiments with different gene constructs in cell transfection assays. Also, data from cell transfection assays are not always consistent with

data from experiments in transgenic mice.

Structure and Functions of the Collagen Triple Helix

The collagen triple helix is fonned from three polypeptide chains that are each coiled into a left-handed helix. The three chains are then wrapped around each

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1995.64:403-434.

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Annu. Rev. Biochem.

COLLAGENS

405

other into a right-handed super-helix so that the final structure is a rope-like

rod 0, 2, 4, 6, 7). The presence of glycine as every third amino acid in the repeating -Gly-X-Y- sequence of each chain is essential, because a larger amino acid will not fit in the center of the triple helix where the three chains

come together. Proline is frequently in the X-position of the -Gly-X-Y- se­

quence and 4-hydroxyproline is frequently in the Y-position. These two amino

acids limit rotation of the polypeptide chains. The triple helix is further stabi­ lized by hydrogen bonds and water bridges, many of which require the presence

of 4-hydroxyproline. The conformation of the triple helix places the side chains

of amino acids in the X- and Y-positions on the surface of the molecule. This

arrangement explains the ability of many collagens to polymerize, since the

multiple clusters of hydrophobic and charged side chains direct self-assembly into precisely ordered structures. The triple helix is relatively rigid. In some contexts, the resistance of the molecule to extension or compression is impor­

tant for the biological function of the protein. In many collagens, the triple helix is interrupted by globular sequences that make the molecule more flex­

ible, but the precise functions of the globular sequences are unknown.

Types of Collagen

For simplicity, the superfamily of collagens (6) can be divided into several

classes on the basis of the polymeric structures they form or related structural

features (see Figure 1): (a) collagens that form fibrils (types I, II, III, V, and

XI), (b) collagens that form network-like structures (the type IV family, and

types VIII and X), (c) collagens that are found on the surface of collagen fibrils and are known as fibril-associated collagens with interrupted triple helices (FACITs that include types IX, XII, XIV, XVI, and XIX), (d) the collagen

that forms beaded filaments (type VI), (e) the collagen that forms anchoring fibrils for basement membranes (type VII), if> collagens with a transmembrane

domain (types XIII and XVII), and (g) the newly discovered types XV and

XVIII

collagens that have been only partially characterized. An additional

group

(h) consists of proteins containing triple-helical domains that have not

been defined as collagens.

FIBRIL-FORMING COLLAGENS All these collagens (types I-III, V, and XI) are

similar in size and in that they contain large triple-helical domains with about 1000 amino acids or 330 -Gly-X-Y- repeats per chain. In addition, they are also first synthesized as larger precursors, and the precursors need to be processed to collagens by cleavage of N-propeptides and C-propeptides by specific proteinases. Finally, they are similar in that they all assemble into cross-striated fibrils in which each molecule is displaced about one-quarter of

its length relative to its nearest neighbor along the axis of the fibril (Figure 1).

Type I is the most abundant coHagen and is found in a variety of tissues. Many

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1995.64:403-434.

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Annu. Rev. Biochem.

406 PROCKOP & KIVIRIKKO

A 1,1I,III,V,XI N-propeplides C-propeptides 1 ! f D (67 nm) staggered. molecues ' " "
A
1,1I,III,V,XI
N-propeplides
C-propeptides
1
!
f
D (67 nm) staggered. molecues
'
" "
"II" "
'� •
'II
II
,
, ---
12345
300nm
IV
200nm
VIII, X
C.
IX, XII, XIV, XVI, XIX
rx
Xli.
GAG
monomer
-----
chain
�I�
?--
-
I !� lilhdl ill
100nm
IifibriJ
100nm
D.
VI
E.
VII
monomer
-
dlmer
lelramer
ttoootI
beaded filament
�chOI=� s:��r
en
agens
1-\'JVllal
macrophage
H.
mannan binding
surfactant
Clq
protein
protein
conglutinin
� J""'-
collagen
c:el
20 nm
tail
mernbtane

Figure 1 Schematic for the structure of various collagens. The figure is modified after the figure presented by Hulmes (6) and reproduced here with permission. The letlers refer to the classifications used in the text Because the protein structures are still unknown, the scheme does not present collagens with a transmembrane domain (types XIII and XVII) and the family of two newly discovered collagens (types XV and XVIII).

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Annu. Rev. Biochem.

COLLAGENS

Table 1

Collagen ty pes and the location of their genes on human chromosomes·

Type Gene Chromosome Ex pression COLlAI 17q2J.3-q22 Most connective tissues COLlA2 7q2I.3-q22
Type
Gene
Chromosome
Ex pression
COLlAI
17q2J.3-q22
Most connective tissues
COLlA2
7q2I.3-q22

II COL2AI 12q13-q14

Cartilage, vitreous humor

407

III eOL3Al

2q24.3-q3l

Extensible connective tissues, e.g. skin, lung, vascular system

IV eOL4Al

13q34

Basement membranes

 

COL4A2

13q34

COL4A3

2q3S-q37

COL4A4

2q3S-q37

eOL4AS

Xq22

COL4A6

Xq22

V COLSAI

9q34.2-q34.3

Tissues containing collagen I,

quantita-

eOL5A2

2q24.3-q3l

tively minor component

COL5A3

VI COL6Al

21q22.3

Most connective tissues

 

eOL6A2

21q22.3

COL6A3

2q37

VII eOL7Al

3p2l

Anchoring fibrils

 

VIII eOL8AI

3qI2-qI3.1

Many tissues, especially endothelium

eOL8A2

Ip32.3-p34.3

IX COL9AI

6q12-q14

Tissues containing collagen II

 

COL9A2

Ip32

COL9A3

X COLl DAI

6q21-q22

Hy pertrophic cartilage

 

XI COLIIAI

Ip21

Tissues containing collagen

II

eOLl IA2

6p2L2

COL2Alb

12q13-q14

XII eOLl 2Al

6

Tissues

containing collagen I

XIII COLl 3AI

IDq22

Many tissues

 

XIV COLl 4AI

Tissues containing collagen I

XV COLl SAI

9q2l-22

Many

tissues

XVI COLl 6AI

Ip34-35

Many

tissues

XVII COLl 7AI

l Oq24.3

Skin hemidcsmosomes

 

XVIII COLl 8AI

21q22.3

Many tissues, es pecially liver and kidney

XIX COLl9AI

6q12-q14

Rhabdomyosarcoma cells

 

a For chromosome locations see References 21,44,75, 102,214. b The a3(XI) chain of type XI collagen is encoded by the same gene as the a I (II) chain of type

II.

of the other fibril-fonning collagens have a more selective tissue distribution (Table 1). Among the new developments concerning fibril-fonning collagens is the

discovery of alternative splicing ofexons in the N-terminal propeptides of type

II (27-29) and type XI collagens (30-32). Because of alternative splicing, the

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Annu. Rev. Biochem.

408 PROCKOP & KlVIRIKKO

coding sequences of an additional exon (27-29) are present in the type II procollagen formed in noncartilaginous tissues early in embryonic develop­

ment (33-37). Another new development concerning fibril-forming collagens is the finding that many fibrils in vivo are composed of two or more different

collagen types (4, 6, 9). An additional discovery is that hybrid molecules

containing chains of both type V and type XI collagens are present in some tissues such as a hybrid molecule containing the a.2(V) chain and the 0.1(XI) chain (4, 6, 9, 38-40). Therefore, type V and type XI collagens can probably

be considered as a single kind of collagen comprised of five different chains

(9), i.e. al(V), a.2(V), a.3(V), a.l(XI) and a.2(XI).

The gene structures of the fibril-forming collagens show a great deal of similarity (12-14). One common feature of the genes is that the major triple­ helical domain of each chain is coded for by 42 exons. Most of the exons are 54 bp and the others are either twice 54, three times 54, or combinations of 45 and 54 bp exons. Also, each exon begins with a complete codon for glycine, and therefore the exon codes for a discrete number of -Gly-X-y- tripeptide units. In addition, the pattern of exon sizes is similar in all the genes and has

been highly conserved throughout evolution. The genes for the 0.2(1) chain of type I collagen and al(Ill) chain of type III collagen contain alternative promoters that code for different polypeptides (41,42). The alternative promoter of the COLlA2 gene is located within intron 2, and the transcript contains a short open reading frame that is out of frame

with the collagen coding sequence (41). Thus, this RNA cannot encode a collagen but may encode a noncollagen polypeptide. The transcript appears early in embryogenesis in tissues derived from neuroectoderm, but at later stages of development, it is found almost exclusively in hyaline cartilage (41). The alternative promoter of the COL3A 1 gene is located in intron 23, and the transcript may encode either a noncollagen polypeptide or a truncated collagen (4 2). This transcript appears tran siently in limb mesenchyme and then de­ creases to low levels in intact cartilage (42). The functions of these alternative transcripts are currently unknown.

NETWORK-FORMING COLLAGENS These collagens include the family of type

IV collagens found in basement membranes and type VIII and X collagens (Figure 1). The collagenous domain of a type IV collagen molecule is longer than in the fibril-forming collagens and consists of about 1400 amino acids in -Gly-X-Y- repeats that are frequently interrupted by short noncollagenous sequences. The N-terminus of a molecule contains a small noncollagenous domain and the C-terminus a major noncollagenous domain of about 230 amino acids (Figure I). The molecules self-assemble to form net-like structures in which monomers associate at the C-termini to form dimers and at the

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COLLAGENS

409

N-termini to form tetramers. In addition to these end-to-end interactions,the triple-helical domains intertwine to fonn supercoiled structures (43-45). Although most of the type IV collagen in basement membranes consist of

a combination of al(IV) and a2(IV) chains, some basement membranes

contain smaller amounts of molecules of a3 (IV) and a4( IV) (46-49) or of a5(IV) and a6(IV) chains (50-56 ) that are similar but not identical. Further variation in the structure of type IV collagens is caused by alternative splicing

of RNA transcripts for the a3(IV) chain (57, 58). Also of interest is that the genes of type IV collagens are found in pairs with head-to-head orientations on different chromosomes so that the promoter regions overlap (Table 1). The al(IV) and a2(IV) chain genes are head-to-head on chromosome 13 (43, 44), the a3 (IV) and a4(IV) chain genes are head-to-head on chromosome

2 (44, 47, 48, 59); and the a5(IV) and a6 (IV) chain genes are head-to-head

on the X chromosome (44,50, 5 1,54, 55, 6 0). The structures of these genes

differ distinctly from those of the fibrillar collagens. Only a few exons are 54 or 45 bp,and many exons coding for the triple-helical domain begin with

a split codon for glycine in which the first G of the codon is in the preceding

exon (12- 14, 44, 6 0).

The two other network-forming collagens,types VIII and X,are very dif­ ferent in structure from type IV but similar to each other (4-7, 9, 6 1). The al(VIII), a2(VIII), and al(X) chains all contain a collagenous sequence of almost the same size and with eight imperfections in similar positions in the -Gly-X- Y- sequences. The genes for these two collagens all contain only three

exons, and almost all of the coding sequences are found in the large third exon (4, 12- 14,6 1). Descemet's membrane. which separates the corneal endothelial

cells

from the stroma. consists of stacks of hexagonal lattices made of type

VIII

collagen (4). Type X collagen is among the most specialized of the

collagens and is

deep-calcifying zone of cartilage (4-7, 6 2). The assembled form of type X collagen resembles the hexagonal lattice of type VIII in De�cemet's membrane (6 2).

synthesized

primarily by hypertrophic chondrocytes in the

collagens (types IX,XII, XIV, XVI,and XIX) do

not fonn fibrils themselves but are found attached to the surfaces of preexisting

fibrils of the fibril-forming collagens (3- 1 1). All these collagens are charac­ terized by short triple-helical domains interrupted by short noncollagenous sequences. The type IX collagen molecule consists of three triple-helical domains and four noncollagenous domains. The protein is commonly found on the surface of fibrils of type n collagen covalently bound to molecules of type II collagen in antiparallel orientation (Figure 1). One unusual feature of collagen IX is that it often occurs as a proteoglycan in which a single glycosaminoglycan

FACIT COLLAGENS

These

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Annu. Rev. Biochem.

410 PROCKOP & KIVIRIKKO

side chain is covalently attached to the second noncollagenous domain of the a2(IX) chain. In ocular and embryonic tissues,type IX collagen occurs in a

form with a short al(IX) chain lacking nearly all of the N-terminal globular

domain. This short al(IX) chain is transcribed from an alternative promoter

located between exons 6 and 7 of the al(IX) gene (63). The expression patterns of the long and short forms seem to be both temporally and spatially regulated. During avian development,the switch in expression from the short form to the

long form occurs at the beginning of chondrogenesis during the early devel­

opment of the vertebral column (64, 65). Type XII and XIV collagens show several structural similarities to type IX collagen,particularly in the C-terminal collagenous domains (3 ,6,8,9,66-70).

These two collagens also contain glycosaminoglycan side chains attached to the large N-terminal globular domain. The RNA transcripts for type XII and

XIV collagens undergo alternative splicing that varies the structures of the

N-terminal globular domain. In the longest form of type XII collagen, the N-terminal globular domain contains 18 fibronectin type III repeats and four repeats homologous to the von Willebrand factor A domain (8, 9). In the longest form of type XIV collagen, the N-terminal domain contains eight and two of these repeats respectively (69). Type XVI collagen (71,72) and the recently discovered type XIX collagen(73,74) also show similarities in struc­ ture to the FACIT collagens and are therefore classified into this subgroup.

The only collagen known to form

beaded filaments is type VI (75) (Figure 1). Each of the three different chains of the protein contains a very short triple-helical domain,and the remainder consists of large N-terminal and C-terrninal globular domains (75). Recently, researchers found that the N-terminal globular region of the a3(VI) chain is much larger than the same region in the other two chains (4-7, 75). The N-terminal and C-terminal globular domains of all three chains contain 200- residue repeats with significant similarities to the A domains of von Willebrand factor. The C-terminal region of the a3(VI) chain also contains three additional domains that show similarities to salivary proteins, to fibronectin type III repeats,and to Kunitz-type protease inhibitors (4-7, 9,75). Several o.2(VI) and a3(VI) chain variants result from alternative splicing of the repetitive noncollagenous subdomains (4-7,9,75,76).

BEADED FILAMENT-FORMING COLLAGEN

COLLAGEN OF ANCHORING FIBRILS Type VII collagen forms anchoring fibrils (Figure 1) that link basement membmnes to anchoring plaques of type IV collagen and laminin in the underlying extmcellular matrix (77-82). The tri­ ple-helical domain of type VII collagen, which is longer than the triple helix of any other collagen, contains 1530 amino acids in -Gly-X-Y- repeats that are interrupted at 19 separate sites (82). The large N-terminal globular domain

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COLLAGENS

411

contains a segment homologous to cartilage matrix protein. The segment is followed by nine fibronectin type III repeats, one segment homologous to von Willebrand factor A domain, and a segment that is cysteine and proline rich. The smaller C-terminal globular domain contains a segment homologous to Kunitz-type protease inhibitors(82). The protein is frrst assembled into anti­ parallel dimers formed by a small overlap at the C-terminal globular ends. The C-terminal globular domains appear to be cleaved during the assembly of dimers and the dimers are stabilized by disulfide bonds. The dimers then associate laterally and in register to become the main constituents of anchoring fibrils. The gene for type VII collagen is about 31 kb and has 118 exons (83). It therefore has more exons than any other known gene.

COLLAGENS WITH A TRANSMEMBRANE DOMAIN

Two recently discovered col­

lagens

contain a transmembrane domain and, therefore, are probably not se­

creted

into the extracellular matrix. Type XIII collagen (11, 84-87) is found

in many tissues. In contrast, type XVII collagen(80,88-9 1) is found primarily in the hemidesmosomes of the skin and is one of the two antigens that produce the autoimmune disease known as bullous pemphigoid. These two collagens are not homologous in structure, but they both contain a single transmembrane N-terminal domain that is apparently cytoplasmic. The remainder of the mole­ cule is extracellular (11,87,90,91). One of the most remarkable features of type XIII collagen is that it undergoes extensive alternative splicing that can generate several hundred forms of the protein (84, 92, 93). The alternative splicing is unique among collagens in that it involves -Gly-X-Y- sequences. How this alternative splicing alters the potential of the protein for folding into a triple helix in which all three chains must be the same length is not clear.

FAMILY OF TYPES XV AND XVIII

The newly

discovered type XV (94-98) and

XVIII

(97, 99- 103) collagens

have a large N-terminal globular domain, a

highly

interrupted triple helix, and a large C-terminal globular domain. Both

collagens also contain several potential attachment sites for serine-linked gly­ cosaminoglycans and asparagine-linked oligosaccharides, observations that suggest these collagens may be extensively glycosylated. Both type XV and type XVIII collagens are found in many tissues, but type XVIII collagen is expressed at much higher levels in the liver. Type XVIII collagen is transcribed from two alternative promoters, and the RNA transcript from one of the

promoters is further modified by alternative splicing within sequences coding for the N-terminal globular domain (103). The longest variant of type XVIII collagen has an N-terminal cysteine-rich sequence that is homologous to three noncollagenous proteins in the family of G-protein coupled receptors ( 103).

"NONCOLLAGEN" COLLAGENS The group of proteins containing collagenous sequences but not defined as collagens includes the subcomponent C lq of

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Annu. Rev. Biochem.

412 PROCKOP & KlVIRlKKO

-- / ,. "" - B ,." -- �� 001-0 J OH t••, =�= ===
--
/ ,.
""
-
B
,."
--
��
001-0
J
OH
t••,
=�=
===
===

Figure 2

Schematic for the biosynthesis of a fibril-forming collagen. (A) Intracellular events that

involve post-translational hydroxylation and glycosylation, association of polypeptide chains, and folding ofthe triple helix. (8) Extracellular events that involve cleavage ofthe N- and C-propeptides,

self-assembly of collagen into fibrils, and cross-linking of the fibrils. Reproduced with permission

from Reference 108.

complement, the tail structure of acetylcholinesterase, pulmonary surfactant proteins SP-A and SP-D, mannan-binding protein, conglutinin, collectin-43, the bacterial enzyme pullanase, and type I and type II macrophage scavenger receptors (6, 11, 104-107). The type I and II macrophage scavenger receptors resemble type XIII and XVII collagens in that they contain a single transmem­ brane domain preceded by a short cytoplasmic N-terminal domain. The rest of the molecule is extracellular (11, 105).

BIOSYNTHESIS

General Features

The fibril-forming collagens are first synthesized as larger precursor molecules

known as procollagens. The intracellular steps in the assembly of a procollagen

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COLLAGENS

413

are (Figure 2) cleavage of signal peptides, hydroxylation of Y-position proline and lysine residues to 4-hydroxyproline and hydroxylysine; hydroxylation of a few X-position proline residues to 3-hydroxyproline, addition of galactose or both galactose and glucose to some of the hydroxylysine residues, addition of a mannose-rich oligosaccharide to one or both of the propeptides, association of the C-terminal propeptides through a process directed by the structure of these domains, and formation of both intrachain and interchain disulfide bonds. After the C-propeptides have associated and each chain has acquired about 100 4-hydroxyproline residues, a nucleus of triple helix forms in the C-terminal region, and the triple helical confonnation is then propagated to the N-terminus in a zipper-like manner (6,7,16,21,108). After secretion of procollagen from fibroblasts, the N-propeptides are cleaved by a procollagen N-proteinase and the C-propeptides by a separate procollagen C-proteinase. The collagen then self-assembles into fibrils. Fi­ nally, lysyl oxidase converts some lysine and hydroxylysine residues to alde­ hyde derivatives that form a complex series of cross-links. The assembly and processing steps of many nonfibrillar collagens are the same, but there are notable exceptions. Many collagens contain N- and/or C-tenninal noncollagenous domains that are not removed and therefore not called propeptides. Several collagens undergo N-glycosylation. Three colla­ gens (types IX, XII, and XIV) are modified by addition of glycosaminoglycan side chains, and two additional collagens (types XV and XVIII) have potential attachment sites for such chains. The triple helices of a few collagens that lack large C-tenninal globular domains (e.g. type XII) may fold by mean s of a mechanism that does not involve formation of a nucleus in the C-terminus

(109).

Intracellular Processing

Recently, analyses of cDNAs provided the complete amino acid sequences for the a. subunit of prolyl 4-hydroxylase from human (110),chick (111), nema­ tode Caenorhabditis elegans (112),and for the subunit from several organ­ isms (113). In addition, complete amino acid sequences have been reported for human (114) and chick (115) lysyl hydroxylase. Prolyl 4-hydroxylase from vertebrates is an ��2 tetramer and lysyl hydroxylase an dimer, but the subunit structure of prolyl 3-hydroxylase is currently unknown (116,117). No significant homology is found between the primary structures of lysyl hydroxy­ lase and the two types of subunits of prolyl 4-hydroxylase in spite of the marked similarities in the catalytic properties between these two enzymes (116, 117). The two catalytic sites in the tetramer of prolyl 4-hydroxylase are located in the a. subunits (113, 116, 117). Even though the enzyme from all the vertebrate sources studied is an ��2 tetramer, cloning and expression of the a. subunit from C. elegans revealed that its prolyl 4-hydroxylase is an 0.13 dimer

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414 PROCKOP & KlVIRlKKO

(112). Also, an isoform of the a subunit of the vertebrate enzyme, defined as

a(U) subunit, was recently discovered (118). Expression studies have demon­ strated that the recombinant enzyme from vertebrates can form both [a(I) h i3 2 and [a(II)hi32 tetramers (118, 119), but whether the recombinant enzyme can also form tetramers that contain both a subunits is currently unknown. There appear to be no major differences in the tissue distribution of the two a subunits (118). Most of the catalytic properties of the [a(I)h� and [a(II)hi32 tetramers are highly similar, but a surprising difference is that poly(lrproline) is a very poor inhibitor of the [a(II)hj32 enzyme (118), whereas it is a highly effective competitive inhibitor of the [a(I)h� enzyme (117). Nucleotide sequencing of the cDNA for the j3 subunit of prolyl 4-hydroxy­ lase indicated that this polypeptide is identical to the enzyme protein disulfide isomerase (POI) (120). Moreover, the j3 subunit has POI activity even when present in the native prolyl4-hydroxylase tetramer (121). POI catalyzes thiol:

disulfide interchange in vitro, leading to net protein disulfide formation, re­ duction, or isomerization depending on the reaction conditions. Researchers regard it as the in vivo catalyst of disulfide bond formation in the biosynthesis of a large number of secreted and cell surface proteins, including collagens

(122, 123).

The POI activity of the PDIIj3 subunit is not directly involved in the hy­ droxylation reaction of prolyl 4-hydroxylase. This finding is based on recent data obtained by expression of a recombinant prolyl 4-hydroxylase tetramer in insect cells (119, 12 4). The PDI1j3 polypeptide has two -Cys-Gly-His-Cys­ sequences that represent two independently acting catalytic sites for the isom­ erase activity (12 4, 12 5). When both these sequences were modified to -Ser­ Gly-His-Cys-, the polypeptide had no PDI activity but still associated with the a subunits to form the �i32 tetramer, and this tetramer proved to be fully active prolyl 4-hydroxylase (12 4). Expression studies have further demonstrated that in the absence of the PDIIj3 subunit, the a subunit forms highly insoluble aggregates (118, 119, 126). Therefore, one function of the PDIIj3 subunits in the prolyl 4-hydroxylase tetramer is to keep the a subunits in a catalytically active, nonaggregated conformation. Recent reports indicate that the cellular PDI1j3 polypeptide may have several additional functions (117, 122, 123, 12 7-12 9). One is to serve as a major cellular thyroid hormone-binding protein in the endoplasmic reticulum. A second function is to act as a chaperone-like polypeptide that nonspecifically binds peptides in the lumen of the rough endoplasmic reticulum. A third function is to serve as the smaller subunit of the microsomal triglyceride transfer protein. Further suggested functions are to serve as a dehydroascorbate reductase (130) and to act as a developmentally regulated retinal protein termed r-cognin (131). The PDI1j3 subunit thus appears to be an unusually versatile

polypeptide that has many biological functions.

415

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COLLAGENS

The two hydroxylysyl glycosyltransferases involved in the biosynthesis of collagens have been extensively characterized (6, 7, 16), but the genes have not yet been cloned. Recent reports also suggest that chain association and folding of type I and IV collagens may involve a specific molecular chaperone protein called Hsp47 or colligin (132-134). Hsp47 binds specifically to type I procollagen and to types I and IV collagens in vitro. Cellular levels of the protein parallel the rates of synthesis of type I or type IV collagen in many experimental situations. Cross-linking studies in intact cells demonstrated association of type I procol­ lagen with Hsp47, and this association was increased when cells were heat shocked or treated with the iron chelator a.,a.'-dipyridyl that effectively inhibits the hydroxylation of proline residues (132). Treatment of cells with antisense oligonucleotides to Hsp47 decreased the rate of synthesis of type I procollagen (134). However, Hsp47 does not bind to type m procollagen, and researchers have not clearly established that it has an essential role in procollagen biosyn­ thesis.

Extracellular Events

Extracellular collagen fibrils are formed by secretion of a soluble procollagen that is then enzymatically processed to an insoluble collagen. The mechanisms by which other collagens are incorporated into an insoluble extracellular matrix are more obscure, since presumably they must be soluble during intracellular assembly. One possible mechanism is that such collagens are secreted as soluble proteins that bind to other macromolecules after secretion to form insoluble heteromolecules. Both the N- and C-propeptides of procollagens must be cleaved by specific proteinases for the proteins to self-assemble into fibrils under physiological conditions. The N-propeptides of both types I and II procollagens are cleaved by the same specific procollagen N-proteinase (6, 7, 16). The N-propeptide of type III procollagen is probably cleaved by a different type III N-proteinase (6, 7, 16). Whether other specific N-proteinases are required to cleave other procollagens such as types V and XI is unclear. Contrary to earlier reports, type I N-proteinase extracted from bovine tissues was recently shown to be the same protein as the better-characterized enzyme from chick embryos (135). Also, researchers recently demonstrated that if type I procollagen is aggregated by addition of polyethylene glycol (136), the rate of cleavage by the C-prote­ inase is increased 10- to 15-fold. The rate of cleavage by the N-proteinase is increased about fourfold. Because the turnover numbers with monomeric type I procollagen are low, it may be that the enzymes in vivo may act on secreted aggregates of procollagen (137). The self-assembly of fibril-forming collagens has been studied for many years by warming and neutralizing solutions of the collagen extracted from

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416 PROCKOP & KIVIRIKKO

tissues with cold acidic buffers (137, 138). More recently, the process was

studied in a system in which pCcoIIagen, a soluble and partially processed

precursor lacking the N-propeptide, is cleaved to collagen by purified procol­ lagen C-proteinase in a physiological buffer and at physiological temperature

ranges (137). Cleavage of pCcollagen to collagen reduces the solubility of the protein by about l000-fold. The resulting collagen reproducibly self-assembles into tightly packed fibrils (137, 139).

One series of experiments was carried out by isolating type I procollagen

and cleaving it with C-proteinase to generate type I pNcoIIagen (137). The

pNcollagen assembled into thin, sheet-like structures that were cross-striated

in longitudinal sections and of a uniform thickness. Mixtures of type I collagen

and pNcollagen copolymerized to form a variety of pleomorphic fibrils. The

results were consistent with the hypothesis that under some circumstances type

I pNcollagen has a biological role in altering the morphology of type I collagen

fibrils (137, 140). Type III pNcollagen also formed true copolymers with type I collagen,and the copolymerization generated fibrils that were thinner than fibrils generated

from type I collagen alone (141). The results were consistent with a model in which type III pNcollagen can regulate the diameter of type I collagen fibrils by coating their surface. However, the effects on fibril diameter required at

least a 1:1 ratio of type III pNcollagen to type I collagen (141). In related experiments, recombinant type II pCcollagen (142,143) was used for fibril assembly by incubation with C-proteinase (144). The kinetics for the

assembly of type II collagen fibrils differed markedly from those for the assembly of type I collagen, and the critical concentration at 37°C was about

50-fold greater. Also, the type II collagen fibrils were relatively thin and formed three-dimensional networks. The results indicated that the differences in primary structure between type II and I collagens are sufficient to explain

many of the characteristic differences in morphology between these two kinds of fibrils seen in tissues (144). The system for generating type I collagen fibrils by enzymic cleavage of type I pCcollagen made it possible to follow the growth of fibrils from the intermediate stages. The first fibrils detected had a blunt end and a pointed tip (145). Initial growth of the fibril was exclusively from the pointed tip. Later, tips appeared on the blunt ends and the fibrils grew in both directions. Both

the initial tips and the later tips were nearly paraboloidal (145). Based on the

observations, a model of fibril growth was developed (146), the essential

features of which were a distinctive structural nucleus that formed at each end of a growing fibril. The growth of the fibril then occurred by propagation of the two structural nuclei. The structural nuclei had similar spiral helical con­ formations, and assembly and propagation of each structural nucleus required just two kinds of specific binding steps (146). Similar studies on fibrils of

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COLLAGENS

417

recombinant type IT collagen demonstrated that the tips were again nearly paraboloidal (147). However, the monomers in the tips of the two types were oriented differently. In tips of type I collagen fibrils, all the monomers were oriented so that the N-termil!i pointed toward the end of the tip (145). In fibrils of type II collagen, all the monomers were oriented so that the C-termini pointed toward the growing tip (147). Recently, Fourier transformed infrared spectroscopy (FTIR) was used to study the lag period of fibrils assembled by neutralizing and warming solutions of collagen extracted with cold acidic buffers (138). The results are consistent with the conclusion that as the tempemture is raised, the triple helix tightens or stiffens but then relaxes again as fibrils are formed (138. 148). The lysyl oxidase that forms cross-links in collagen fibrils is a highly insoluble copper-containing protein (149). Complete cDNA-derived amino acid sequences have now been reported for the enzyme from several sources (150-153). The enzyme was identical to a tumor suppressor protein known as rrg (154. 155). Earlier work had demonstrated that lysyl oxidase activity is markedly low in the culture medium of many malignantly transformed human cell lines (156), and recently, these cells were also found to have very low levels of lysyl oxidase mRNA (157).

Potentials for Inhibiting Fibrosis

Normal wound healing involves the formation of scars and fibrous tissue that largely consist of collagen fibrils. Although moderate degrees of fibrous tissue are beneficial in wound repair, fibrous material often accumulates in excessive amounts and impairs the normal function of the affected tissue. Such excessive accumulation of collagen becomes an important event in scarring of the skin following burns or traumatic injury and in fibrosis of the liver, lungs, and kidneys following injury to these organs. Therefore, there has been consider­ able interest in agents that can inhibit or modulate collagen synthesis in fibrotic diseases. Potential target sites for inhibiting collagen synthesis include tran­ scription of the genes, translation of the mRNAs, and some of the unique post-translational enzymes involved in the biosynthesis of the protein. Recent studies have demonstrated that synthesis of type I collagen can be specifically inhibited in cell culture by the use of antisense oligonucleotides (158-160). However, the degree of inhibition obtained is highly variable and rarely exceeds 50% (158, 1 59). In related experiments. an antisense gene to human type I collagen in which only the 3'-half was inverted was shown to be highly effective in inhibiting collagen synthesis in transgenic mice express­ ing an internally deleted human COLlAI minigene (161). The results raised the possibility that chimeric gene constructs that contain intron sequences and in which only part of the gene is inverted may be particularly effective as antisense genes that can inhibit collagen synthesis in fibrotic conditions. How-

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418 PROCKOP & KlVIRIKKO

ever, both the antisense-oligonucleotide strategy and the antisense-gene strat­ egy appear to present considerable problems in the delivery of the agents in ways that will be effective in inhibiting fibrosis in vivo. Several of the post-translational enzymes appear to be attractive targets for specific inhibition because they are unique to collagen biosynthesis. These include prolyl 4-hydroxylase, procollagen C-proteinase, and perhaps also lysyl hydroxylase and lysyl oxidase. Numerous compounds are now known that inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate (116, 117). For example, pyridine 2,4-dicarboxylate inhibited prolyl 4-hydroxylase with a Kj of 2 J,1M. The problem of cell membrane permeability was in part overcome by the design of lipophilic proinhibitors that were converted to the active inhibitors in­ tracellularly (21, 117, 162). One such derivative inhibits hepatic collagen accumulation in two models of liver fibrosis in rats (21, 162). The recent success in expression of an active recombinant human prolyl 4-hydroxylase in insect cells (119) should make it possible to define the critical structural features of the enzyme by site-directed mutagenesis (119, 163) and to produce adequate amounts of the enzyme for crystallization so that more effective inhibitors can be designed. Procollagen C-proteinase is another attractive target for inhibition of fibro­ sis. Most of the available evidence suggests that procollagen cannot participate in fibril assembly unless the C-propeptide is specifically cleaved from the precursor (6, 7, 16, 137). The only challenge to this proposal comes from experiments in which recombinant procollagen was synthesized with a muta­ tion at the cleavage site so that the protein was not cleaved by C-proteinase. Some of the protein synthesized in cell culture was cleaved by nonspecific proteases (164; J Bateman, personal communication). However, such nonspe­ cific cleavage is unlikely to generate collagen that is assembled into normal fibrils. Initial studies suggested that basic amino acids and peptides may specifically inhibit C-proteinase (see 108), but the development of more ef­ fective agents such as peptidomimetics is still in the early stages. Several attempts have been made to develop inhibitors for Iysyl hydroxylase. Minoxidil and many of its derivatives have the surprising effect of reducing both lysyl hydroxylase activity (165) and the mRNA (166, 167) in cultured cells. Their mechanism of action is unknown. Also, whether inhibition of lysine hydroxylation will in itself be effective in inhibiting fibrosis is unclear. One of the first targets explored for inhibition of fibrosis was lysyl oxidase. Aminopropionitrile has long been known to be a suicide inhibitor of the enzyme (149). Recently, several derivatives were developed that are even more effective (149). However, whether an inhibition of lysyl oxidase will prevent fibrosis is unclear, because the cross-linking of collagens occurs long after fibril assembly.

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COLLAGENS

419

MUTATIONS IN MEN AND MICE

Mutations in Patients

TYPE I COLLAGEN Almost 200 different mutations have now been charac­ terized in the eOLlAI and eOLlA2 genes that code for proal(I) and proa2(1) chains of type I procollagen (2, 17-22,25; for details, see 18). Most of these

mutations have been identified in patients with osteogenesis imperfecta (01), but they are also found in patients with related disorders (Table 2). OJ is characterized by brittle bones but also involves other tissues rich in type I collagen so as to produce blue sclerae, abnormal teeth, thin skin, weak tendons, and hearing loss. In the most severe forms, bones and other tissues are so fragile that death occurs in utero or shortly after birth. In more moderate forms, the disease is not lethal, but the patients have repeated fractures after.

minor trauma that may lead to permanent deformities of limbs and other bony structures.

Table 2 Diseases caused by mutations in collagen genes or deficiencies in

the activities of post-translational enzymes of collagen synthesis

of post-translational enzymes of collagen synthesis Gene or enzyme Disease COLlAl; COLlA2 Osteogenesis

Gene or enzyme

Disease

COLlAl; COLlA2

Osteogenesis imperfecta

 

Osteoporosis' Ehlers -Danlos syndrome type

VIlA, VIIB

COL2 Al

Several chondrodysplasias Osteoarthritis' Ehlers-Danlos syndrome type IV

COL3Al

C0L4 A3; COL4A4 COL4 AS

Aortic aneurysms' Alport syndrome, autosomally inherited forms Alport syndrome, X-linked form Alport syndrome with diffuse esophageal

COL4AS and COL4 A6

le iomyomatosis,

X-linked

COL7 Al

Epidermolysis bullosa, dystrophic forms Osteoarthritisb

COL9Al

COL9A2

Multiple epiphyseal dysplasiac

 

COLlO AI

Schmid metaphyseal chondrodysplasia Stickler syndrome, nonocular forme Ehlers-Danlos syndrome type VI

eOLl lA2

Lysyl hydroxylase

Type

IN-proteinase

Ehlers-Danlos syndrome type VIle Occipital hom syndromed Menkes syndromed

Lysyl oxidase

In a subset of patients. b Demonstrated only in transgenic mice.

C Demonstrated only by genetic linkage.

d Secondary to an abnormality in copper metabolism.

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420 PROCKOP & KIVIRIKKO

Essentially all patients with OI have mutations in type I collagen . Most of the mutations are single base substitutions that convert a codon for an obligate glycine in the repeating -Gly-X-Y- sequence of the triple helix to a codon for an amino acid with a bulkier side chain (2, 17-22). Other mutations include deletions, insertions, RNA splicing defects, and null alleles. The null alleles primarily cause the mild type I variant of OI (168). The mutations inactivating the alleles have been difficult to define, but four patients were recently found to have a premature translation termination codon that decreased the cytoplas­ mic level of the mRNA (J Korkko, P Paassilta, J Zhuang, H Kuivaniemi, L Ala-Kokko, et al, in preparation). Mutations that cause synthesis of structurally altered proo. chains of type I procollagen generally cause more severe phenotypes than null alleles (2, 17- 22). One of two molecular mechanisms are usually involved. Some substitu­ tions for obligate glycines interrupt the zipper-like folding of the triple helix and generate unfolded procollagen that fi rst ac cumulates in fibroblasts and is then degraded. The effects of the mutations are amplified because both the

normal and mutated chains present in the same molecule are degraded in a

process referred to as procollagen suicide or a dominant negative effect (17, 18, 108). The effects of other glycine substitutions are explained by their consequences on the nucleated growth of collagen fibrils. One such mutation was a heterozygous substitution of cysteine for glycine at position 748 of the 0.1(1) chain that introduced a flexible kink into the triple helix (2, 18, 169). Studies on fibril fo rmation in vitro (see biosynthesis) demonstrated that mole­ cules with the cysteine kink copolymerized into fibrils with the normal mole­ cules, but the presence of the kinked molecules delayed fibril formation, reduced the total amount of collagen incorporated into the fibrils, and drasti­ cally altered the morphology of the fibrils (18, 1 38, 17 0) (Figure 3). Other

glycine substitutions that do not affect folding have similar effects on fibril

assembly (1 71-173).

In 01, bones are fragile in part because of a marked reduction in bone mass (osteopenia). Mutations in type I collagen have also been found in a few patients who have little evidence of OI but who have osteopenia and fractures characteristic of osteoporosis (18, 20, 21, 25, 174, 175). A recent survey suggested that 1-3% of patients with osteoporosis have a mutation in either the eOL lAI or eOLlA2 gene (175). Some mutations in the type I collagen genes produce a disease known as the type VII variant of EDS (Table 2), a syndrome characterized by joint hypermohility and skin abnormalities (18, 21 , 23 , 25). The disease is caused

by a failure to cleave the N-propeptide fr om type I procollagen. The persistence

of the N-propeptide on the molecule drastically alters fibril formation so that the fibrils become thin and highly irregular in cross-section. The mutations causing EDS VII are either RNA splicing mutations that eliminate the amino

irregular in cross-section. The mutations causing EDS VII are either RNA splicing mutations that eliminate the

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421

= / ;' ;' ;' ;' / / B ;' / ) . Procollagen Suicide
=
/
;'
;'
;'
;'
/
/
B
;'
/
)
.
Procollagen
Suicide

Abno rma l Fib r II s

Schematic of how mutations such as glycine substitutions alter the biosynthesis of

collagen. (A) Illustration of a glycine substitution that prevents the zipper-like folding of the triple

helix and leads to degradation of both normal and abnormal pro« chains by a procollagen suicide or dominant negative mechani sm. (8) Illustration of a glycine substitution that does not interfere with the folding of the triple helix but produ ces a conformational change such as a kinkin the protein. (Modified and reproduced with permission from Reference 108.)

Fig ure 3

acids of the cleavage site for the N-propeptide (subtypes VIlA and VIIB) or mutations that decrease the activity of the cleaving enzyme, procollagen N­ proteinase (subtype VIle).

Over 50 different mutations in the COL2A1 gene have

been shown to cause a heterogeneous group of disorders of cartilage that are known as chondrodysplasias and are characterized by short-limbed dwarfism

TYPE II COLLAGEN

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422 PROCKOP & KIVIRIKKO

and skeletal deformities (18, 21, 24, 176-182). The mutations include amino

acid substitutions, deletions, insertions, RNA splicing defects, and stop codons

for premature termination of translation. All five stop codons so-far charac­

terized have been identified in the single phenotype of the Stickler syndrome that involves vitreous degeneration and retinal detachment in addition to de­

generation of joint cartilage (21, 183-187). Mutations in type II collagen (fable 2) are also found in about 2% of patients

with early-onset familial osteoarthritis (177). One mutation is a substitution of cysteine for arginine at amino acid position 519 of the 0.1(11) chain, that has

now been found in four apparently unrelated families with severe, early-onset

osteoarthritis and mild chondrodysplasia (179, 188, 189). Two additional mu­

tations in patients with a similar disease phenotype are a serine-for-glycine substitution at position 0.1-274 (190) and at 0.1-976 (179). In addition, muta­

tions in other genes for collagens such as types IX and XI (Table 1) may be

found as predisposing factors for osteoarthritis in some families (see Mutations

in Transgenic Mice, below).

TYPE III COLLAGEN About 50 different mutations in the COL3A1 gene have

been found in patients with EDS IV (18, 21, 23, 191, 192), the most severe form of EDS that can cause sudden death from rupture of large arteries and

other hollow organs in addition to skin and joint changes (23, 25). The muta­ tions include glycine substitutions, deletions, RNA splicing defects and null alleles (18, 21, 191, 192). Mutations in the COL3Al gene have also been found

in a subset of patients who have arterial aneurysms but who exhibit little (193) or no evidence (18, 21, 194-196) of other connective tissue manifestations. Mutations in the COL3A1 gene may also be a predisposing facto. for intrac­

ranial aneurysms (18, 21, 197), but they appear to be a rare cause of this disease

(198).

TYPE IV COLLAGEN No mutations have been identified in the genes eOUA l

and eOUA2 that encode the two major a. chains of type IV collagen, but mutations have been found in the genes coding for the minor type IV collagen

polypeptides (fable 2). The Alport syndrome is a progressive heritable kidney disease characterized by hematuria caused by structural changes in the

glomerular basement membrane. This disease is also associated with hearing loss and ocular lesions. The gene coding for the a5(IV) chain was mapped to

the locus for the X-linked form of the Alport syndrome (50, 51), and sub­ sequently, more than 50 different mutations in the eOUA5 gene were found

in families with this disorder (21, 44, 199, 2(0). The mutations include amino acid substitutions, large deletions, and gene rearrangements such as inversions, insertions, and duplications. Although the Alport syndrome is primarily X­ linked, autosomally inherited forms also exist, and recently, heterozygous

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COLLAGENS

423

mutations in both the COIAA3 and COIAA4 genes were characterized in autosomally inherited forms of the disease (44, 20 1). Deletions involving the head-to-head 5'-ends of both the COIAA5 and COIAA6 genes have been fo und in several patients (54, 202) who have the Alport syndrome together with diffuse esophageal leiomyomatosis, a rare syndrome characteri zed by proliferation of smooth muscle cells in the esopha­ gus, tracheobronchial tree, and the female genital trac t (44).

TYPE VII COLLAGEN About 20 mutations in the COL7Al ge ne have been found in patients with the dystrophic form of epidermolysis bullosa. a disease characterized by severe blistering and scarring of the skin from minor trauma (78-8 1 , 203-206). As a consequence of these mutations, the anchoring fibrils that link the basement membrane to the anchoring plaques in the skin are either reduced in amount or completely absent (203). Mutations in the COL7Al gene were fo und in both the dominantly and recessively inherited forms of the disease (78-8 1). The mutations include amino acid substitutions, an insertion­ deletion, and premature translation termination codons (8 1, 203-206).

TYPE X COLLAGEN More than 10 different mutations have now been charac­ terized in type X collagen (207-213) in patients with Schmidt metaphyseal chondrodysplasia, a disease that is characterized by shortening of limbs and bowing of legs aggravated by walking. The mutations include amino acid substitutions, deletions, and premature translation termination codons. All the mutations so far characterized alter the structure ofC-terminal noncollagenous domain of the polypeptide, an observation suggesting that the mutant chains are unable to associate to fo rm triple-helical molecules .

OTH ER COLLAGENS Genetic linkage was fo und between the COL9A2 ge ne locus (214) and multiple epiphyseal dysplasia (EDM2). Also, genetic linkage was found between the COLl IA2 gene locus and a non-ocular form of the Stickler syndrome (2 15). Since correct expres sion of collagen genes appears to be essential for the structural integrity of many tissues, mutations in more than 30 different col­ lagen genes (Table 1) can probably produce disease phenotypes. Therefore, research in this area is still at a very early stage (20). Also, similar disease phenotypes are probably produced by mutations in genes for other matrix proteins, since several diseases of cartilage have been linked to loci that do not contain any known collagen genes (18, 26).

POST-TRANSLATIONAL ENZYMES A defic iency of lysyl hydroxylase is found in most but not all families with EDS VI (2 1 , 23, 108), a disease characterized by hyperextensible skin and joints, scoliosis, and ocular fragility. One mutation

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424 PROCKOP & KIVIRIKKO

in the gene for the enzyme was a homozygous duplication of seven exons that

appears to be caused by a recombination of Alu sequences (216, 2 1 7). The

same mutation was fo und in several apparen tly unrelated fa milies (2 16-218). Additional mutations include a homozygous translation termination codon

(219) and several amino acid substitutions (220). EDS vile is caused by a defic iency in type I procollagen N-proteinase (22 1, 222), but no mutations in the genes have been characterized so far.

Deficiencies of Iysyl oxidase, a copper-containing protein, are seen in two rare and severe X-linked recessive diseases, the occipital horn syndrome and Menkes syndrome (2 1, 108. 223). The diseases are caused by defects in copper metabolism and lead to secondary defects in the cross-linking of collagen, but the mechanisms producing a deficiency of Iysyl oxidase are unclear. Skin

fibroblasts from patients with these diseases contain and secrete reduced amounts of the lysyl oxidase protein (21 . 224), and recently, two (157, 224) out of three (1 57. 224, 225) studies reported that these cells also contain

reduced amounts of Iysyl oxidase mRNA. These observations suggest that the abnormality in copper metabolism somehow influences the synthesis or sta­

bility of the mRNA for Iysyl oxidase.

Mutations in Transgenic Mice

Transgenic mice are particularly useful for studying matrix protei ns because most of the proteins are large, insoluble. and difficult to test for function. They are also useful for studying the consequences of disease-causing mutations in matrix genes, since the mutations affect many tissues that cannot be examined fully in patients. Experiments have been carried out in which mutated collagen

genes were randomly inserted in transgenic mice to produce dominant negative

effects, and more recently. a few experiments were carried out involving knock-out of col lagen gene s. In some instances. the re sults from these two

types of experiments have been complementary . In others they are apparently contradictory. The first experiments with transgenic mice used a retrovirus infection of mouse embryos. By chance. the retrovirus was inserted in the first intron of the COLlAI gene and prevented expression of the gene in most tissues (see226). Homozygous mice died in utero with liver necrosis and bleeding. Heterozygous mice survived and had decreased collagen content, decreased mechanical strength of bones. and hearing loss (226). No fractures were de­ tected. Subsequently. transgenic mice were prepared that expressed a mutated COLl AI gene in which a cysteine codon was substituted for an obligate glycine (227). Four of seven founder mice had some of the phenotypic features of 01 such as poor mineral ization in most bony structures. Again. however. no fractures were demonstrated and no breeding lines of the transgenic mice could be developed. In contrast. extensive fractures were observed in several

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lines oftransgenic mice expressing a mini-gene version ofthe human COLIAI gene in which exons 6 to 46 were deleted in-frame to cause synthesis of shortened proal (I) chains (228). Mice expressing high levels of the mini-gene protein developed a lethal phenotype of extensive fractures because the short­ ened proa chains produced procollagen suicide or a dominant negative effect through protein depletion (228). Mice expressing lower levels developed a milder phenotype resembling human osteoporosis (229, 230). Extensive breed­ ing of the transgenic mice in the inbred line demonstrated marked phenotypic variability and incomplete penetrance that apparently is an inherent property of expression of mutated collagen genes (230). In related experiments, a naturally occurring recessive mutation that produced bowed and brittle bones in mice was shown to be a single base deletion in the C-propeptide that caused synthesis of proa2 chains that could not associate with proal chains (23 1 ). As a result, the only type I procollagen synthesized was homotrimers of proal(I) chains. In experiments with the COL2AI gene, transgenic mice expressing an internally deleted version of the human gene developed a phenotype similar to some human chondrodysplasias with dwarfism, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone (232). At the same time, similar phenotypes of severe chondrodysplasias were seen in transgenic mice expressing mouse COL2Al genes mutated either by substitution of a cysteine codon for glycine at amino acid position 85 in the al (lI) chain (233) or by a deletion that eliminated amino acids 4-18 of the a l (II) chain (234). In older mice from some of the same lines, the evidence of chondrodysplasias was less marked, and the most striking features were degenerative changes of articular cartilage similar to osteoarthritis (235, 236). A surprising finding was that over-expression of a normal mouse COL2A l gene in transgenic mice produced abnormally thick collagen fibrils in cartilage, apparently because of an imbalance in the amounts of different collagens being synthesized in the tissue (237). Transgenic mice over-expressing the normal gene developed a chondrodysplasia Transgenic mice were prepared that expressed a COL3A 1 gene in which a methionine codon was substituted for lysine at amino acid 939, the cross-link­ ing site in the triple-helical region of the protein (238, 239). The phenotype was mild, but pregnant females apparently developed uterine dysfunction (238). The same mice had morphologic changes in healing dermal wounds, but no drastic consequences occurred on wound repair (239). Transgenic mice expressing a mutated COL9Al gene (240) and transgenic mice in which the gene was inactivated (241) de veloped early onset osteoarthri­ tis. The results suggested, therefore, that a subset of patients with osteoarthritis may have mutations in type IX collagen. In transgenic mouse experiments with the COLIOAI gene, the results with

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426 PROCKOP & KIVIRIKKO

dominant negative experiments and gene inactivation experiments were dif­

ferent. Transgenic mice expressing constructs of the chicken gene with an

in-frame deletion in the central triple-helical domain of the collagen (242) developed skeletal abnormalities and a deficiency of leukocytes similar to the Schmidt type of human metaphyseal chondrodysplasia. The observations in transgenic mice (242) largely provided the basis for defining mutations in the same gene that cause Schmidt phenotypes in patients (see above). However, mice with homozygous inactivation of the COLlOAI gene did not develop any apparent phenotype (243). The initial explanation of these conflicting data was that mutations producing a dominant negative effect were more severe in terms of phenotype than mutations that inactivated the gene. Subsequently, researchers found that mice harboring a 68-arnino acid deletion from the triple-helical domain of the type X collagen gene did not develop any gross abnormalities of the skeleton (244). At the moment, there is no simple way to resolve these conflicting observations. Transgenic mice expres sing an internally deleted COL l IA2 gene developed a mild phenotype with a short snout, prominent forehead, shortened limbs, and shortened tail (245). Also, DNA linkage to the locus for COL l IAI was found in cho mice that have a naturally occurring autosomal recessive chondrodys­ plasia (246). The o. l (XI) chain was ab sent fro m tissues, and the collagen fibrils in cartilage from the mice were unusually thick.

Potentials for Gene Therapy

Researchers have conducted preliminary experiments to test the potentials of gene therapy for either controlling coll agen deposition in fibrotic conditions or rescuing the phenotypes produced by mutated genes . As indicated above, oligonucleotides partially inhibited expression of collagen genes in cell culture experi ments ( 1 58-1 60). A re lated approach showed that an antisense gene to the human COLlAI gene is effective in transgenic mice (161 ). The experi­ ments were carried out with mice that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the proo.I (1) chain of human type I procollagen. The mice were bred to transgenic mice that expressed an antisense gene that was similar to the internally deleted human COL lAI mini-gene, but the 3'-half of the gene was inverted so as to code for hybrid sense and antisense RNA. In mice that inherited both genes, the incidence of lethal fragile bone phenotype was reduced from 92 to 27% (161 ), and there was a corresponding decrease in the tissue levels of the human mini-proo.l (I) chains. The experiment may have succeeded where similar

experiments with antisense cDNAs failed in the past, because the transcript from the antisense gene contained inverted intron sequences and, therefore, interacted with the sense transcript in the nucleus. In related experiments, normal mice were subjected to lethal body irradiation

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and then received stromal cells of bone marrow from a transgenic mouse

containing a DNA marker consisting of a human COLlAl mini-gene. After five months, the donor cells were fo und to account for 3-5% of the cells in bone marrow, spleen, bone, cartilage, and lung (247). Also, the human

COLlAl mini-gene was expressed in bone. Because stromal cells from bone

marrow are readily expanded in culture, they may be a useful source of long-lasting precursor cells for gene therapy of bone and cartilage diseases.

Techniques were also developed for site-directed insertion of an exogenous

collagen gene into an endogenous gene locus (248). In stable transfection experiments with the human tumor cell line known as HT-1080, a construct

containi ng a short 5'-fragment from the CO LlAI gene li nked to 30 kb of the

COL2A l gene was fo und to be in serted at a high frequency into both alleles

of the endogenous COLlAI gene (248). Transfected cells with the targeted

insertion expressed relatively high levels of the exogenous gene. The results

suggested that it may be possible to target insertion of exogenous genes into predetermined loci of collagen genes. The targeted insertion has the advantage

that it provides controlled expression of the exogenous genes and avoids the potential dangers of random insertions.

SUMMARY

Collagens are defined as proteins that: (a) contain several repeats ofthe amino acid sequence -Gly-X-Y- in which the X-position is frequently proline and the

V-position is frequently 4-hydroxyproline and (b) have the potential for three chains with such repeated sequences to fold into a characteristic triple helix.

At least 19 proteins and more than 30 gene products are now formally defined as collagens. An addi tional 10 proteins have collagen-like domains. Therefore, the number of proteins classified within this super-family is rapidly expanding. The most abundant collagens form extracellular fibrils, but others form net­

work-like structures. Still others bind to the surfaces of collagen fibrils, form beaded-filaments, serve as anchoring fibrils for the skin, and are transmem­ brane proteins. The exon-intron patterns of most of the collagen genes are

complex. Most of the genes are widely distributed in the genome, but three pairs of genes are located in a unique head-to-head arrangement with overlap­

ping promoters.

Recently, several of the eight highly specific posttranslational enzymes involved in collagen biosynthesis were cloned. The 13 subunit of prolyl 4-hy­

droxylase is identical to the enzyme protein disulfide isomerase and appears to have several other distinct functions. Experiments with recombinant pro­ collagens that are the soluble precursors of fibril-forming collagens have

confmned previous indications that collagens self-assemble into fibrils by the classical mechanism of nucleation and propagation. The results also indicated

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428 PROCKOP & KIVIRIKKO

that the differences in primary structure between the two most abundant fibril­ forming collagens (types I and II) are sufficient to explain many of the char­

acteristic differences in morphology between these two kinds of fibrils. There has been increasing interest in the possibility that the unique post­

translational enzymes involved in collagen biosynthesis offer attractive targets for specifically inhibiting the excessive deposition of collagen fibrils that occurs in scars and in the fibrotic response of injury seen in most tissues. Inhibition of prolyl 4-hydroxylase and procollagen C-proteinase appear to be

particularly attractive strategies. The important roles of collagens in biology have been illustrated by the recent demonstrations that over 400 mutations in 6 different collagen types cause a variety of human diseases. Mutations in the type I collagen genes can produce defec ts of bones and related tissues that range from lethal osteogenesis

imperfecta to osteoporosis. Mutations in the gene for type n collagen can produce cartilage disorders ranging from lethal chondrodysplasias to early-on­ set osteoarthritis. Mutations in the gene for the type X collagen, which is expressed in hypertrophic chondrocytes, also produce chondrodysplasias, and

genes for two additional collagens of cartilage have been linked to similar phenotypes. Mutations in type III collagen produce defects of blood vessels and other tissues that range from a severe form of the Ehlers-Danlos syndrome to familial aortic aneurysms. Mutations in several polypeptide chains of the type IV collagens of basement membranes produce the renal disease known as the Alport syndrome that may be associated with diffu se esophageal leiomyomatosis. Mutations in the type VII collagen that forms anchoring fibri ls for basement membranes cause severe blistering and scarring of the skin. Mutations in other collagens are also likely to produce human diseases, but some diseases with similar phenotypes are clearly linked to noncollagen genes. Experiments with transgenic mice have been particularly useful for defining

the structure and function of collagens because the proteins are large, insoluble, and difficult to test for function. In addit ion, transgenic mice have been useful in demonstrating the consequences of disease-causing mutations in collagen gene s, si nce the mutations affect many tissues that cannot be examined fully in patients. Experiments with mutated genes for type I collagen reproduced the phenotypes of osteogenesis imperfec ta and osteoporosis. Experiments with mutated genes for type II collagen reproduced the phenotypes of severe chon­ drodysplasias and osteoarthritis. Similar experiments with the type III collagen gene produced only a mild phenotype. Experiments with the type IX gene produced early onset osteoarthritis. Experiments with a dominant negative version of the type X gene produced a phenotype of a specific chondrodys­ plasia, but a knock-out experiment with the same gene produced no apparent phenotype. In exploring potentials for gene therapy, oligonucleotides were shown to

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partially inhibit expression of collagen genes in cell culture. In stable cell­ transfection experiments, a hybrid gene containing a 5' fragment of the type I collagen gene was found to be inserted into both alleles of the normal locus of the type I gene at a high frequency. The results suggested it may be possible to target insertion of exogenous genes into predetermined loci of collagen genes. A phenotype of severe osteogenesis impetfecta in transgenic mice expressing a mutated human type I collagen gene was partially rescued by breeding these mice to other transgenic mice expressing an antisense version of the same human gene.

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