Ascorbate requirement for hydroxylation and secretion

of procollagen: relationship to inhibition

of collagen synthesis in scurvy1’2
Beverly Peterkofsky

ABSTRACT Vitamin C deficiency is associated with defec- independent processes, with hydroxylation occurring as a post-
tive connective tissue, particularly in wound healing. Ascorbate translational modification of the protein (7, 8). Therefore, the
is required for hydroxylation of proline residues in procollagen results derived from hypro analyses became ambiguous because
and hydroxyproline stabilizes the collagen triple helical structure. it was not clear whether collagen polypeptide synthesis or proline
Consequently, ascorbate stimulates procollagen secretion. hydroxylation had been affected.
However, collagen synthesis in ascorbate-deficient guinea pigs Collagen is composed mainly of repeating glycine tripeptides
is decreased with only moderate effects on proline hydroxylation. with the sequence -gly-x-y-, where y is frequently hypro. Bacterial
Proteoglycan synthesis, which does not require ascorbate, also collagenase cleaves collagen at the N terminal ofthe gly residue
is decreased and both effects are correlated with the extent of whether or not the proline in the y position is hydroxylated.
weight loss during scurvy. Fasting, with ascorbate supplemen- This specificity was exploited to devise an assay for measuring
tation, produces similar effects. Both functions are inhibited in collagen synthesis by using purified bacterial collagenase (9, 10).
cells cultured in sera from either scorbutic or starved guinea pigs By incubating cells or tissues with proline labeled uniformly
and inhibition is reversed with insulin-like growth factor (IGF)- with carbon-l4 and tritium in the 4 position, it became possible
I. The inhibitor appears to consist of two IGF-binding proteins to measure collagen synthesis and proline hydroxylation inde-
induced during vitamin C deficiency and starving and may be pendently but simultaneously (10). The tritium atom is displaced
responsible for in vivo inhibition of collagen and proteoglycan during hydroxylation so that the percentage of proline residues
synthesis. Am J C/in Nutr 1991 ;54: 11 35S-40S. in collagen that are hydroxylated can be calculated from the
ratio of ‘4C to 3H in collagenase-released peptides.
KEY WORDS Collagen, proteoglycan, scurvy, ascorbate,
Ascorbate as a cofactorfor prolyl hydroxylase
insulin-like growth factors
A requirement for ascorbate in proline hydroxylation was es-
tablished in a cell-free system composed of microsomes con-
Introduction
taming unhydroxylated collagen and prolyl hydroxylase (8). The
Defective connective tissue in vitamin C deficiency enzyme was subsequently isolated from a number of sources
and additional requirements for a-ketoglutarate and ferrous ion
From the earliest descriptions of scurvy, the disease caused
were found (1 1). Lysyl hydroxylase, which catalyzes the for-
by vitamin C deficiency, it was evident that there were effects
mation of hydroxylysine in collagen, has similar requirements
on connective tissues, especially in healing wounds (1). The his-
(1 1). Prolyl hydroxylase is a tetramer composed of a and fi sub-
tory ofexperiments that led to the conclusion that collagen me-
units that have been sequenced ( 1 2). The enzymatic mechanism
tabolism was affected by the disease and that formation of hypro
proposed by Hanauske-Abel and Gunzler (13) suggests that hy-
(hydroxyproline), an imino acid found almost uniquely in col-
droxylation of proline occurs through a reactive iron-oxygen
lagen, might be involved was reviewed extensively (2-5). complex, the ferryl ion, and that ascorbate is required to reduce
Guinea pigs, like humans, cannot synthesize ascorbic acid
Fe3 formed during the reaction.
and thus have been used as a model for studying the effects of
vitamin C deficiency (2). Early biochemical studies on the effects The role ofhypro in stabilizing the collagen triple helical
of scurvy on collagen synthesis frequently were carried out with structure
repair tissue from carrageenin-induced granulomas or polyvinyl There are at least 14 types of collagen but each consists of
sponge implants in guinea pigs (5). Collagen synthesis was mea- three subunits with their collagenous domains in a triple helical
sured by analyzing hypro in collagen extracted from the repair configuration (14-16). For fibrillar collagens, preprocollagen is
tissue or by measuring the incorporation of radioactive proline
into hypro in minces ofthe tissues. Because hypro concentrations
were decreased in the wound tissue ofvitamin C-deficient guinea I From the Laboratory of Biochemistry, National Cancer Institute,
pigs, it was concluded that collagen synthesis was decreased. It Bethesda, MD.
2 Address reprint requests to B Peterkofsky, Building 37 Room 4C-
was later discovered, however, that synthesis of the collagen
polypeptide and hydroxylation ofprolyl residues in collagen were 18, National Cancer Institute, Bethesda, MD 20892.

Am J C/in Nuir 1991;54: 1 l35S-40S. Printed in USA. © 1991 American Society for Clinical Nutrition ll35S

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 1 136S PETERKOFSKY

synthesized on polysomes bound to the rough endoplasmic re- secretion is specific since secretion of noncollagenous proteins
ticulum (RER) and after cleavage of the signal peptide, the re- was not affected (Fig 2, C).
suIting procollagen is transported through the Golgi pathway
and secreted (1 5, 16). The propeptides at the N and C terminals Use ofascorbate in cell culture
of the collagen chains are enzymatically cleaved and the triple In species that do not require ascorbate, its synthesis occurs
helical collagen molecule is incorporated into the extracellular in the liver (mammals) or kidney (birds), so that connective
matrix. tissue cells in culture, regardless of the species of origin, are
The steps in procollagen biosynthesis involved in the hydrox- incapable of synthesis. In addition, fetal calf serum processed
ylation reactions are presented in Figure 1 . Prolyl hydroxylase for cell culture contains almost no ascorbate (23), so that as-
is localized in the cisternae of the RER ( I 5, 17) whereas lysyl corbate must be added to cultures to obtain proline hydroxyl-
hydroxylase activity is distributed between the membrane and ation. Ascorbate generally is not required for cell growth and
cisternae ( 1 8). Hypro plays a critical role in stabilizing the triple can produce cytotoxic effects when concentrations from 0.05 to
helical structure of the collagenous domain of procollagen (15), 0.30 mmol/L are added to cells at low densities(24). Its instability
which appears to be preferentially secreted. Therefore as hy- in culture media (23) can be overcome by repeated additions or
droxylation occurs, the helix forms and rapid secretion takes by the use of ascorbate-2-phosphate (25), although cells must
place. Inhibition of hypro formation, either by omission of as- be able to cleave the phosphate group to use the compound, and
corbate from cultured cells (19) or by the addition of a,a’-di- phosphatases in serum must be heat inactivated. There are several
pyridyl, an iron chelator, to cell or organ cultures (15, 19), leads cell lines that do not require ascorbate for proline hydroxylation
to slower secretion. Microsomal transport systems for ascorbate (23, 26) because they contain a microsomal protein capable of
(20) and a-ketoglutarate (21) have been described. The ascorbate acting as a reducing cofactor (27). The active sequence of the
uptake system is iron dependent (20). Interchain disulfide bonds protein consists of cysteinyl-cysteine (28).
between the carboxyl propeptides are formed in the absence of
hydroxylation but the chains are denatured (15, 22). An example Relationship ofproline hydroxy/ation to regulation of collagen
of the effects of ascorbate on secretion of procollagen is shown production in vivo
in Figure 2. Human-skin fibroblasts were labeled with radioactive In most short-term experiments in cell culture systems, as-
proline and the rates ofprocollagen synthesis and secretion were corbate had no effect on collagen production (Fig 2) (19). In
measured. The rate ofcollagen synthesis relative to total protein contrast, several studies showed a direct induction of collagen
synthesis was unaffected by ascorbate (Fig 2, A) but without gene expression by ascorbate after long-term exposures (24-72
ascorbate the initial rate of secretion was much slower (Fig 2, h) ofchick-embryo tendon cells(29) and human-skin fibroblasts
B). With time, however, the rate ofsecretion of unhydroxylated to ascorbate (30). In the human-fibroblast system, the stimulation
procollagen increased. The effect of ascorbate on procollagen appears to be related to lipid peroxidation rather than to the

ARG
HIS RAPID
LH GLY
LYS
SECRETION

PRO
GLY

ePRO PRO
GLY

NH2 [

aa-DIPYRIDYL OR

SLOW
SECRETION

FIG I. Posttranslational hydroxylation of procollagen. PH, LH: prolyl and lysyl hydroxylases.

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 COLLAGEN SYNTHESIS IN SCURVY ll37S

free diet for “-‘2 wk and then begin to lose weight at variable
rates. Death usually occurs after ‘-4 wk on the experimental
Is regime. Our initial studies in calvarial bone showed that collagen
Is
Is production decreased after ‘-2 wk but there was only a moderate
.c
decrease in proline hydroxylation (33). The decrease in collagen
C
>1 production was due to decreased synthesis and not to an increase
U)
C in degradation. Although there was little correlation of decreased
Is collagen synthesis with the effect of scurvy on hydroxylation,
Is there was a direct correlation with the extent of weight loss.
0 Guinea pigs that received vitamin C but had food withheld so
0 that they lost weight at rates equivalent to those ofthe scorbutics
100 showed equivalent decreases in collagen synthesis (33). Similar
results were obtained from studies with articular and costal car-
80 tilage (34, 35). Proline hydroxylation in articular cartilage was
C
0 especially resistant to ascorbate deficiency (34), with only a 15%
60 decrease after 3 wk on the ascorbate-free diet, whereas collagen
U
Is synthesis was decreased to 50% of the control values.
Cl) 40 In cartilage of scorbutic guinea pigs, proteoglycan synthesis
C was decreased coordinately with collagen synthesis and there
Is
20 was a direct correlation between the rates of synthesis of both
0 components and the rate ofweight loss(35). Similar correlations
0 were found in cartilage of guinea pigs acutely starved but sup-
60 plemented with vitamin C (35, 39). Because there is no known
role for ascorbate in the synthesis ofproteoglycans, these results
ae 50
supported the conclusion that the effect ofvitamin C deficiency
- 40 on collagen synthesis was independent
of the role of ascorbate
in proline hydroxylation. collagen synthesis in scor-
Decreased
Is
U butic cartilage was accompanied by a decrease in the steady-
Is
Cl) 20 state concentration of type II procollagen mRNA (34) whereas
decreased proteoglycan synthesis may be caused by a decrease
2; io in the activity of galactosyl transferase, an enzyme involved in
z
initiation of the chondroitin sulfate chain (40). On the basis of
0 2 4 6 these studies, we (41) proposed that vitamin C deficiency and
fasting had similar consequences on the synthesis of extracellular
Time (hr)
matrix components.
FIG 2. Procollagen synthesis and secretion in human skin fibroblasts. The fact that collagen synthesis decreased in a number of
Cultures were labeled with [5-3H]proline without(-)orwith (+)ascorbate connective tissues in scorbutic and starved guinea pigs (33-35,
and analyzed at the times indicated as described previously (10). Secretion
39) suggested that a circulating factor might be involved in the
is expressed as the percentage of total radioactive protein that is in the
regulation (41). Sara from guinea pigs that were either fasted
medium. A, relative rate ofcollagen synthesis; B, procollagen secretion;
C, noncollagenous protein (NCP) secretion. (ascorbate supplemented) or placed on a vitamin C-deficient

role of ascorbate in proline hydroxylation (3 1, 32). Because of

the ambiguity of results from cell culture and early in vivo cx-
periments, we examined the relationship between proline hy-
droxylation and collagen metabolism in vivo in nonrepair con-
nective tissues by using the techniques we developed to measure
these indices independently (10).

Results

Weanling guinea pigs were placed on an ascorbate-free diet

and control animals were supplemented orally with vitamin C
(33-35). After 1 wk on an ascorbate-free
diet, the concentration
ofascorbate in most tissues is reduced to 10% ofnormal values ‘
8 10 12 14 16 18 20 22 24 26 28
(33, 36, 37). Even after 4 wk, however, the ascorbate concen- Days on diet
tration in bone from scorbutic guinea pig remained at 4% of the
FIG 3. Guinea pig growth curves. Strain 2 female guinea pigs (130-
control value (33). Typical growth curves for control and scor- 140 g) were placed on an ascorbate-free diet and either supplemented
butic guinea pigs are shown in Figure 3. As shown in other daily with 50 mg ascorbate orally (control) or not (scorbutic). The two
studies (35, 36, 38), guinea pigs grow normally on the ascorbate- curves for scorbutics represent different patterns of weight loss.

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 1 138S PETERKOFSKY

diet for periods that resulted in various degrees of weight loss, NORMAL SERUM
were analyzed for several hormones and growth factors (42).
Similar profiles were obtained for sera from starved guinea pigs
and vitamin C-deficient animals that had lost approximately HMW
the same amount ofbody weight (> 20%) as the starved animals
(Table I). There were changes in concentrations of insulin-like COMPLEX
growth factor (IGF)-I, thyroxin, growth hormone, and cortisol.
In scorbutic animals the extent of the changes was dependent
on the amount ofweight lost (42).
Exposure of cultured chick-embryo chondrocytes or human
fibroblasts to starved or scorbutic guinea pig sera reduced collagen 10 F-I
and proteoglycan synthesis to 40-50% of the values obtained
with cells cultured in normal guinea pig serum (43, 44). Dc- RECEPTOR
creased synthesis was not due to lack ofa stimulating factor but
rather to the presence ofan inhibitor (44). Starved and scorbutic
guinea pig sera also lacked mitogenic activity for 3T3 cells (42),
and this effect also was due to the presence of an inhibitor. In FASTED OR SCORBUTIC SERA
all cases, inhibition could be reversed by the addition of IGF-I
(42, 44). IGF-I mediates the effects ofgrowth hormone in vivo,
is required for DNA synthesis, and stimulates the synthesis of
proteoglycans and collagen (45). Almost all of the circulating HMW
IGFs are bound in a complex containing a binding and a non- COMPLEX
binding subunit with a total mass of approximately 150 kDa
(46). There also are smaller IGF-binding proteins in sera with
unoccupied binding sites (46). 10 F-I

JJ,
The inhibitor of IGF-I activity in sera from starved and scor-
butic guinea pigs appears to consist oftwo low-molecular-weight
IGF-binding proteins with unsaturated binding sites. In Figure IGF-I
4 a model is presented to explain the mechanism by which IGF-
binding proteins could inhibit 1GF-I-dependent functions in RECEPTOR LMW
cultured cells. In cells cultured with serum from normal guinea 10 F-B P
pigs, IGF-I would be derived from the high-molecular-weight FIG 4. Proposed model for inhibition ofIGF-I activity by unsaturated
binding-protein complex (HMW complex) by equilibrium be- IGF-binding proteins.
tween bound and free forms. Free IGF-I binds to its cell receptor
to stimulate cell proliferation and cell-specific functions such as
proteoglycan and collagen synthesis. The concentration of low-
molecular-weight binding proteins (LMW IGF-BP) in normal binding sites and thus can compete with the cell receptor for
serum is low but is increased at least 10-fold in starved and free IGF-I and inhibit its functions.
scorbutic guinea pig sera (47). When sera were fractionated on
a gel-filtration column, binding-protein activity and the ability Discussion
of fractions to inhibit binding of [‘25IJIGF-I to its receptor on
3T3 cells coincided (47). The binding proteins have unoccupied Events relevant to extracellular matrix synthesis that occur in
guinea pigs placed on a vitamin C-free diet are summarized in
Table 2. The time-course has been divided into two phases, each
TABLE 1 ‘-2 wks long and separated by the point at which animals stop
Profiles of circulating hormones and growth factors in scorbutic and growing and begin to lose weight. The cause
major event ofthis
starved guinea pigs* in the course ofscurvy is not clear. Several reports indicate weight
loss results from decreased food intake (36, 38), which suggests
Hormone Starved (4 d) Scorbutic (26-28 d)
that ascorbate is involved in functions regulating appetite. It
EGF ND No change also is clear that control guinea pigs that are fed the same amount
IGF-II No change No change of food consumed by scorbutics do not lose as much weight as
Cortisol t t the scorbutics lose (36, 38). Therefore, digestion or metabolism
Growth hormone t offood may be inefficient in vitamin C-deficient animals. Early
Thyroxine in phase I, ascorbate concentrations in blood and tissues fall
IGF-I rapidly. Another early event is a decrease in alkaline phosphatase
C EGF, epidermal growth factor, IGF, insulin-like growth factor. ND, activity in serum and bone tissue (48, 49), which suggests that
there is an effect on osteoblast function during phase I; this has
not determined. The scorbutic guinea pigs had lost > 20% oftheir body
weight from the time of their highest weight until they were killed. The not been demonstrated. Our recent work suggests that in non-
arrows indicate whether the concentration decreased or increased as repair connective tissues, the effects of vitamin C deficiency on
compared with concentrations in normal guinea pig serum. Details are collagen and proteoglycan synthesis occur during phase II and
given in reference 42. may be related to induction of a circulating inhibitor of IGF-I

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 COLLAGEN SYNTHESIS IN SCURVY 1 1 39S

TABLE 2
Consequences of vitamin C deficiency in guinea pigs: events relevant to the regulation of extracellular matrix components6

Phase 1 -+ Phase II

Normal growth Appetite loss or inefficient metabolism Growth stops, weight loss
Vitamin C concentrations decreased Altered concentrations of circulating hormones
Alkaline phosphatase decreased Increase in unsaturated circulating LMW IGFBPs
Increase in circulating inhibitor of IGF-I activity
Decreased collagen and proteoglycan synthesis

C C C S C

Moderately decreased pro hydroxylation in collagen

Poor wound healing

* Each phase is -2 wk.

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