Eur. J. Biochem. 214. 29.

'S-3()4
© FEBS

Novel hirudin variants from the leech Hirudinaria manillensis

Amino acid sequence, cDNA cloning and genomic organization
Emanuela SCACHERI'. Gianpaolo NITTI'. Barbara VALSASINA'. Gaetano ORSINl'. Carlo VISCO'. Monica FERRERA'.
Roy T. SAWYER' and Paolo SARMIENTOS'
' Biotechnology Department. Farmitalia Carlo Erba. Nerviano. Milan. Italy
- Biopharm Ltd. Hendy. Dyfed. Wales
(Received November.'). IW2/February 24. IW.^) - EJB 92 I.'i69/1

Novel hirudin variants isolated from the leech Hirudinaria manillensis. a leech more specialized
for matntnalian parasitism, are described. Isolation of antithrombin polypeptides was performed by
ion-exchange chrotrtatographies followed by an affinity chromatography step on immobilized throm-
bin. The tnajor active component, antithrombin polypeptide peak 2 (HM2). and a second polypep-
tide. named HMl. were purified to homogeneity and their complete amino acid sequences were
detennined. The protein structure ofthe two hirudin variants include 64 amino acids with 6 cysteine
residues at highly conserved positions. Comparison of the amino acid sequences of HMl and HM2
with other known hirudins shows differences tnainly in the central part and in the C-terminal region
of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C-
terminal portion of the molecule which is replaced by aspartic acid.
Polytiierase chain reaction cloning techniques were used to isolate and characterize the cDNAs
and detertnine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding
for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20
residues constitute the signal peptide required for extracellular secretion. The leader sequence ap-
pears to be highly conserved for both isoforms and shares a complete similarity with the partial
hirudin variant 2 (HV2) signal peptide sequence previously reported.
The HMl and HM2 gene fragments show the presence of four exons: the first one corresponding
to a 2()-atnino-acid signal peptide while the other three exons share the full primary structure of the
antithrombin polypeptides.
HM2 was also efficiently produced in rccotnbinant E.scherichia coli by expressing a periplasmic
construction containing the synthetic gene.

Hirudin. the most potent thrombin inhibitor known, is a at multiple sites: the N-terminal globular domain binds near
small pt>lype.ptide discovered over UK) years ago in the saliva the active site of throtnbin while the extended C-terminal
of the medicinal leech Hirudo medicinalis 111. It was first segment, which is abundant in acidic residues and includes a
characterized biochetiiically by Markwardt |2. .^| and its sullated tyrosine in position 6.^. tnakes both ionic and hy-
structure was lately elucidated by Dodt et al. |4|. It is a sin- drophobic interactions with the fibrinogen binding site of
gle-chain polypeptide composed of 65 amino acids, including thrombin |5 —8|.
6 cysteine residues, and a molecular tiiass of about 7(XX) Da. Several isoforms of hirudin have been isolated from the
Hirudin produces its action by binding directly to thrombin medicinal leech |9. 10| showing a high degree of similarity
to each other. Recently we focused our attention on the isola-
Correspondeinc lo E. Seacheri. Farmitalia Carlo Erba. Bioteeh- tion of hirudin-like polypeptides frotn different species of
nology Department. Laboratory of Molecular Biology. Via Papa Gio- leeches and in particular frotn the Asian butfalo leech Hiru-
vanni XXIII 23. I 2(X)14 Nerviano. Milano. Italy j,-,,^,,.,-^, manillensis. This species belongs to the same order
AMrni!i!ionl' MMl. HM2. HM.^. antithron.bin polypeptide Arynchobdellida as H. n.eJicinalis but is significantly more
peaks I 2 and ^ from Hmutinaria ma,,ilhnsis: HVI. himdin variant spec.ahzed tor mammaltan parastttsm 1111.
1: HV2. hirudin variant 2: PCR. polymerase chain reaction: RP- Whtle thc productton ot sutttctcnt atnounts ot htrudtn and
HPLC. reverse-phase high-perfortiiance liquid chromatography: isotbmis by molecular biology techniques has now allowed
Pth-Xaa. phenylthiohydantoin derivative of amino acid: ATU. the study of its characteristics and properties 11 2|. still little
antithromhin unit: TIU. throtiitiin inhibitor unit: Ptc-Xaa. phenylthi- |s known about the tiiolecular biology of this molecule and
ocarbamoyl derivative of amino acid: RACE, rapid amplitication of j,j. variants. In 1986, Harvey et al. isolated the cDNA encixJ-
cDNA ends. , , , , ui u J u u itig hirudin variant 2 (HV2) 11.^1 and this remains the onlv
Noiv. The novel nucleotide sequence data published here have , , .. ,. ,. nxiA
heen deposited with the EMBt. sequence data bank and are available J^'^, available on the htrud.n mRNA. „,, , , ^,,,
under the accession number X7278.S and X72786. The novel amino •" this work two novel htrudm vanants. HMl and HM2.
acid sequence data have also been deposited with the EMBL se- that exhtbtted antithrotiibin activtty. were puritied to homo-
quence data bank. geneity frotn extracts ofthe head of H. manillensis and their
2%
amino acid sequences were determined with microanalytical
methods. On the basis of structural information, we applied
the polymerase chain reaction (PCR) technique to isolate the
cDNAs coding tor the hirudin-like polypeptides HMl and
HM2. In addition, by cloning the genomic fragments of HMl
and H1VI2. we have elucidated for the first time the gene
organization of hirudin-like polypeptides. Fully active re-
combinant HM2 was also produced in Esctierichia coti cells
following transformation with a synthetic gene and using a
periplasmic expression system.
MATERIALS AND METHODS
Leeches
Starved H. manillcnsis leeches were provided by Bio-
pharm UK. Leech heads were dissected out from bodies,
washed in ".> M NaCl. and quickly fro/en in liquid nitrogen
prior to storage at -8()"'C.
Isolation of antithrombin polypeptides
Crude extracts were prepared from the head part of the
leeches essentially as described previously |14|. The purifi-
cation process was monitored by assaying the antithrombin
activity.
Lyophili/ed extracts were dissolved in 20 mM ammo-
nium formate pH 3.0. and applied to a Mono S HR 5t5 col-
umn (Pharmacia) equilibrated in 0.1 M ammonium formate
pH VO. Elution was carried out increasing the pH to 4.5
with a linear gradient in 60 min. Pooled active fractions were
lyophili/ed. dissolved in 20 mM ammonium formate pH 7.0
and loaded onto a Mono Q HR 515 column (Pharmacia). Step
elution was carried out with 0.15. 0.4 and 0.8 M NaCl in
amtTionium formate buffer.
Bovine thrombin (Sigma) was further purified according
to a described procedure \\5\ and immobili/ed to activated
Sepharose CL 6B following manufacturer's instructions. The
active pool from Mono Q chromatography was adjusted to
pH K.3 and applied to a small column (1.7 ml) of immobi-
li/ed thrombin equilibrated in 50 mM Tris/HCl pH S.3. The
column was washed with lO-ml aliquots of Tris buffer. Tris/
3 M NaCl and Tris buffer.
Bound material was then eluted with 25 mM 4-amino-
ben/amidine and desalted against 10 mM sodium phosphate
pH 7.0 on a Smart system using a fast desalting PC 3.2/10
column (Pharmacia). The last step of purification was per-
lonned by reverse-phase high-performance liquid chroma-
tography (RP-HPLC) using a C, Vydac column (4.6X
250 mm. 5 ^m). Antithrombin polypeptide isoforms were
separated with 20 mM sodium phosphate pH 7.5 (eluent A)
and 95'/r acetonitrile (eluent B) in a linear gradient from
5'/' to 25'/f B in 30 min. Fractions corresponding to purified
isoforms were concentrated under vacuum.
Structural analysis of antithrombin polypeptides
Purified antithrombin polypeptides (5-.5()ng) were re-
duced with a 50-fold molar excess of dithiothreitol/6 M gua-
nidine in Tris/HCl pH K.5 and .V-pyridylethylated by reacting
with 4-vinylpyridine |16|.
Derivati/cd or intact polypeptides were digested with
trypsin (treated with tosylphenylalanine chloromethane) or
with Siaphxiococcus aureus VH protease (Sigma) |17|. The
resulting peptide mixtures were lyophili/ed and subjected to
peptide mapping by RP-HPLC on a column of Waters |iBon-
dapak C,K (3.9 X 300 mm. 10
N-terminal .sequence analysis
N-terminal sequence analysis of intact or .S-pyridylethy-
lated antithrombin polypeptides and tragments generated
thereof by en/ymatic cleavages was performed by automated
Edman degradation using a pulsed liquid-phase sequencer
model 477A with an on-line analy/er model 12()A (Applied
Biosystems) tor the detection of phenylthiohydantoin deriva-
tives of amino acids (Pth-Xaa). Standard manutacturers' pro-
grams were used with minor moditications.
C-terminal sequence analysis
C-terminal sequence analysis was performed atter time-
course digestion of intact antithrombin polypeptides [5 —
10|ag) with carboxypeptidase P (Boehringer Mannheim) as
previt)usly described |181.
Biochemical analyse.s
Antithrombin activity was assayed by two //; virro tests.
The first one was based on the inhibition of thrombin-cata-
ly/ed conversion of fibrinogen to an insoluble clot and the
results were expressed in antithrombin units (ATU) 119|. The
other test measured the inhibition of thrombin-mediated re-
lease of /j-nitroanilide from the chromogenic substrate S-
2238 (Kabi-Vitrutn) and the results were expressed in throm-
bin inhibition units (TIU) |2()|. A working standard of throm-
bin solution was calibrated against a specimen of First In-
ternational Standard of human thrombin (National Institute
tor Biological Standards and Controls. South Mimms. UK).
The amino acid compositions of polypeptide preparations
were determined by analysis of the phenylthiocarbamoyl de-
rivatives of amino acids (Ptc-Xaa) atter vapor-phase hydroly-
sis with 6 M HCl. Automated Ptc-Xaa analysis was per-
tbrmed on an Applied Biosystems amino acid derivati/cr
model 420 A equipped with an on-line HPLC. Protein con-
centration was determined with the Bradtbrd procedure |2t |
using bovine serum albumin as a standard.
Isolation and characterization of RNA and DNA
Total cellular RNA was prepared trom leech heads essen-
tially as described by Harvey et al. |13|.
DNA was extracted from tro/en //. nidnittensis leeches
according to the Applied Biosystems protocol tor DNA/RNA
extractor.
For Southern analysis 10 ng genomic DNA was used tor
each digestion tbilowing incubation conditions recom-
mended by the manufacturers. Restriction fragments were
separated on a 0.8% agarose gel and transterred to a nitrocel-
lulose tilter (Millipore) essentially as described by Sambrook
et al. |22|. Standard pre-hybridi/ation and hybridi/ation con-
ditions were used |22|; the ''P-labeled probe was prepared
by the random prime method using the multiprime DNA la-
belling kit (Amersham). The filter was washed to a final
stringency of NaCl/Cit (NaCI/Cit is 0.15 M NaCl. 0.015 M
trisodium citrate. pH 7.0) 0.1% SDS, at 65"C tbr 30 min
and autoradiographcd at -8()"C' using Hypcrliliii MP
(Amersham).

cDNA .synthe.sis
Thc reverse transcription reaction was carried out in a
40-^1 volume as follows: 10 (ig total RNA from leech heads
was tnixed with 1 (ig oligo(dT) pritner. 8 )il 5 niM dNTP mix
and 8 (tl reverse transcriptase buffer (250 niM Tris/HCl pH
8.3. 300 mM KCl. .50 mM MgCI,. 5 mM dithiothreitol).
heated to 65°C for 2 min and quick chilled on ice. 10 U
RNasin (Protnega) and 20 U avian myeloblastosis virus re-
verse transcriptase (Boehringer Mannheim) were added, and
the tube was incubated at 42°C for 2 h. The reaction mixture
was phcnol/chloroform-cxtt acted, isopropanol-precipitated
and lesuspended in 60 (.tl sterile distilled water.
Oligonucleotide.s
Oligonucleotides primers were synthesized on an Applied
Biosystctiis model 38()B DNA synthesizer. Primers utilized
for amplification of cDNA sequences were the following:
dT17 adaptor primer. 5' GACTCGAGTCGACATCGATTT-
TTTTTTTTTTTTTT3': adaptor primer. 5' GACTCGAGTC-
GACATCG X: (irimer 3 - 8 . 5' ATCGAAGCTTA(TC)AC-
(CAT)GA(TC)TG(TC)ACNGA 3'; primer 5 6 - 5 2 . 5' CTAA-
QQAICCTTC(TC)TC(GA)AA(GA)TCNCC 3'; primer 3 2 -
37 5' ATCGGAATTCAGTTCTGGAAATCAGTGCGT 3':
primer 5'RT. 5' CTAAGAATTCTTCGCAACTTATATGC-
GTT 3': primer 6 4 - 6 0 . 5' ATCGGAATTCTTAATTCAAT-
ATATCTTCAT 3'. Primer for DNA amplification was
primer signal peptide 1 - 7 . 5' AGTCGACAAATGTTCTCT-
CTCAAGTTGTTC 3'. The restriction sites added at the .5'
end of each primer to facilitate cloning and sequencing of
the amplification product are underlined. N denotes a mixture
of all four nucleotides.
PCR amplifications
To obtain the complete sequence of HMl and HM2
cDNAs. three rounds of PCR amplification were perfonned.
Eirst round
5 (il reverse-transcribed RNA was amplified in a mixture
containing 10 (.tl lOX PCR buffer (Cetus/Perkin Elmer). 2 (tl
0.1 M MgCl.. UntI dNTP tiiix (1.25 tiiM each dNTP) and
500 pmol degenerated primers 3 - 8 and 56-52. Prior to add-
ing 2.5 U Tciq polymerase (Cctus/Perkin-Elmer) the leaction
mixture was heated at 95°C for 5 min and then allowed to
cool at 70"C. Amplification was achieved with a cycle ot 1-
min denaUiration at 94"C. 2-min annealing at 6()°C and 2.5-
min polymera.se extension at 72 X" for a total of 30 cycles,
with a final 7-min 72"C extension step, using a Cetu.s/Perkin-
Elmcr DNA thetmal eyelet.
297
Third round
To clone the 5' end of HMl and HM2 cDNAs. we also
utilized the RACE protocol. Reverse transcription of head
RNA was performed using the 5'RT primer, as previously
described. The reaction mixture was then isopropanol-precip-
itated and the first-strand cDNA products were polyadeny-
lated at their 3' ends using terminal deoxynucleotidyltrans-
ferase (BRL) |23|. 10 (il ofthe polyadenylated products was
amplified using 10 pmol dT17 adaptor primer. 25 pmol adap-
tor primer and 25 ptnol second-gene-specific primer ( 6 4 -
60) upstream to the first used for transcription: 10 (il of the
amplification product was then reamplified in the same con-
ditions.
PCR amplification on DNA
2 (ig leech DNA was amplified in the presence of
100 pmol 1 - 7 signal peptide pritner and the 6 4 - 6 0 primer
as described (24]. Each cycle of PCR included DNA denatur-
ation at 94°C for 30 s followed by primer annealing for 15 s
and polymerase extension at 72°C for 30 s. The annealing
temperature was gradually increased: for the first five cycles
45°C. 50°C for other five cycles and then 55°C for 25 cy-
cles.
Analysis of PCR products
Amplified products were analyzed on \.59r agarose gel.
phenol-purified and ethanol-precipitated. After cleavage at
restriction sites present in each primer, they were subcloned
into pUC vector, using standard procedures |22|. Sequences
were obtained on both strands using the sequenase kit
(United States Biochemicals).
For Southern blot analysis. 15 (.il amplification products
was subjected to electrophoresis on a 1.5% agarose gel and
subsequently transfeired onto a Hybond-N ' tnembrane (Am-
ersham) as described in the manufacturers' protiKoI. The '-P-
labeled probe was prepared as described before. After hy-
bridization, conducted using standard conditions |221. the
membrane was washed at increasing stringency up to
0.05 X NaCl/Cit. 0.1 9r SDS at 65 "C.
Recombinant DNA techniques
All DNA manipulations were carried out essentially as
described by Satnbrook et al. |22|. Oligonucleotides were
synthesized using an automated DNA synthesizer tnodel
38()B (Applied Biosystems) and purified by polyacrylamide
gel electrophoresis. Oligonucleotides were subcloned in a
M13 vector and their sequences were verified using the
seqtienase kit (United States Biochetnicals).

Second round Expres.sion and purification of recombinant HM2

Amplification of cDNA 3' ends was achieved followitig A 4-1 fennentation of recotnbinant E. coli strain B were
the method of rapid amplification of cDNA ends (RACE) carried out in a tettacycline-containing mediutn. for 8 h at
|23|. IOO pmol gene-specific pritner (32-37) was used to- 37°C. Bacterial cells were harvested by low-speed centrifu-
gether with 100 pmol dT17 adaptor primer to amplify 5 ^i\ gation and stored at -2()''C utitil used. The periplastnic
first-strand cDNA. as previously described. 5 (.il (if this am- content was released by stirring the whole cell paste in Ibr-
plified product was subjected to a second amplification in mate buffer pH 3. Raw extracts were first purified on a
the same conditions but using a more stringent cycle: 1.5- Sepharose-S column at pH 3.1 with an increasing salt gradi-
min denaturation at 94"C followed by 2.5-min annealing ex- ent ( 0 - 1 M NaCl). After adjustment at pH 7. the recombi-
tension at 72"C. Alter 30 cycles, the reaction tnixture was nant HM2-containing fractions were loaded on a Sepharose
extended for 7 min at 72 X\ 6B column chelating Cu ' ions (Phartnacia). previously
298

0.3 - Table 1. Amino acid composition of H. manillensis antithrom-

2 bins. Cys was determined as .S'-pyridylethylated derivative; n.d.. not
s determined.

Amino HM-1 HM-2

0.2 -- acid
t
hydro- sequence hydro- sequence
lysis lysis
residues/molecule
I "•' li'
Asx 7.0 10 8.0 9
a i
Glx 9.6 9 11.2 11
Ser 3.4 4 6.6 7
Gly 9.2 11 K.2 .S
1 i l l , ,
His 0.6 1 0.8 1
t 0 t 5 2 0 2 5
Arg 0.12 0 0.2 0
time (min) Thr 2.7 4 •4
Fig. 1. RP-HPLC purification of H. manillensis antithrombin Ala 0.21 0 0.3 0
polypeptides. Following thrombin-Sepharose chromatography, the Pro 3.0 3 2.5 3.
active pool was purified on a C4 Vydac column eluted with a sodium Tyr 2.1 2 L7 1
phosphate/acetonitrile system at neutral pH as described in Materials Val 2.3 3 3.3 4
and Methixls. The three indicated peaks have been respectively des- Met L3 1 0.3 I)
ignated HMl. HM2 and HM3. Cys 5.6 6 5.5 6
lie 1.9 2 L« '2
Leu 2.9 3 3.0 3
Phe 1.0 1 0.9 1
equilibrated in 50 mM sodium phosphate pH 7 containing Lys 2.8 4 2.5
0.5 M NaCl. The metal-chelating column was stepwisc Trp n.d. 0 0
n.d.
eluted with phosphate buffer of decreasing pH. HM2 was
eluted in the pH 4 phosphate step. Buffer exchange in ammo-
nium bicarbonate was carried out on a Sephadex G-25 col-
umn and solid HM2 was obtained by lyophilization of vola- quence: -(Glu. Asp)-Ile-Leu-Asn-COOH. Amino acid analy-
tile bicarbonate buffer. The purity of the final HM2 prepara- sis showed a few significant difterences between HMl and
tion was assayed by RP-HPLC and N-terminal sequence HM2 composition (Table 1). especially the presence of one
analysis. methionine residue in HMl.
These preliminary structural analyses revealed about
70% of the whole amino acid sequence of bolb isoforms (as
RESULTS expected for a birudin variant of about 60 amino acids) and
Purification and amino acid sequencing indicated that the most marked differences concerned the
of antithrombin polypeptides from H. manillensis central part of the two polypeptides between residues 20 —
40.
Antitbrombin polypeptides were isolated from crude ex- Determination of tbe complete amino acid sequence was
tracts obtained from tbe head parts of H. manillensis leeches. achieved by peptide mapping analysis of HMl and HM2 per-
Atler acetone/acid extraction of the crude material, two steps formed after reduction and alkylation of cysteine residues
ot ion-exchange chromatography were carried out. First the witb 4-vinyl-pyridine. Two different enzytnatic treatments,
crude extract was purified by cation exchange on a Mono S cleavage with trypsin and .V. aureus V8 protease, were em-
column eluted by increasing the pH from 3 to 4.5. The active ployed in order to generate the proper overlapping fragments.
p(H)l was then chromatographed on a Mono Q column using Fig. 2 (in Supplement) shows the RP-HPLC profiles of the
a stepwise gradient of NaCl. The active purified material was tryptic maps obtained tor the two polypeptides. The peptides
subjected to affinity chromatography on thrombin-Sepharose isolated in sufficient yields from both tryptic and V8 protease
in (irder to isolate only ttiose polypeptides which specifically digestion were subjected to N-terminal sequence analysis and
bind to thrombin. The antithrombin polypeptides were fur- the complete amino acid sequences of HMl and HM2 were
ther tractionated by RP-HPLC on a C, Vydac column eluted finally determined (Fig. 3). Several features can be illustrated
at neutral pH. This step was essential to separate the isoforms by the comparison of the five sequences shown in Fig. 3. (a)
from each other. Three main active peaks (labeled HMl. The sequences of HMl and HM2 differ in 10 atnino acid
HM2 and HM3) were obtained by RP-HPLC of the material residues all occurring in the central part of the polypeptides
purified by affinity chromatography (Fig. 1). (between positions 19 and 39) and theretbrc share a sim-
HMl and HM2 were then obtained in homogeneous form ilarity of about 84%; their sitnilarity with hirudin variant 1
atter re-chromatography and subjected to structural analyses. (HVl) is 64% and 70%. respectively, (b) The six cysteine
When direct N-terminal sequencing of 1-nmol samples of residues are found at the same position for all of the five
alkylated HMl and HM2 was performed, the tirst 42 and variants, (c) There are no potential sites tor sulfation in the
45 amino acid residues, respectively, were unambiguously C-terminal part of HMl and HM2. as they lack the tyrosine
identified, with the exception of cycle 43 of HM2 analysis residue present in HVl at position 63.
which gave no identifiable Pth-Xaa. The threonine residue at position 43 of HM2 could not
Time-course hydrolyses of the native proteins with car- be directly identified, since it gave no identifiable derivative
boxypeptida.se P resulted in the release of the same residues for the corresponding cycle during N-terminal sequencing
from the C-terminus. suggesting the following C-terminal se- and its presence was interred by amino acid analysis of tryp-
 299

HVl Vnl Val Tyr Thr Asp Cys Thr Glu Sor Gly Gin Asn Leu Cys Leu Cys Glu Gly Ser Asn
HV2 He Thr Tyr Thr Asp Cys Thr Giu Ser Gly Gin Asn Leu Cys Leu Cys Glu Gly Ser Asn
HV3 lie Thr Tyr Thr Asp Cys Thr Glu Ser Gly Cln Asn Leu Cys Leu Cys Glu Gly Ser Asn
HMl Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys I.eu Cys Val Gly Gly Asn
HM2 Val Spr Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys Val Gly Ser Asn

Val Cys Gly Gin Gly Asn Lys Cys He Leu Gly Ser Asp Gly Glu Lys Asn Gin Cys Val
Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser Asn Gly Lys Gly Asn Gin Cys Val
Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser Gin Gly Lys Asp Asn Gin Cys Val
Leu Cys Gly Gly Gly Lys His Cys Glu Met Asp Gly Ser Gly Asn Lys Cys Val
Val Cys Gly Glu Gly Lys Asn Cys Gin Leu Ser Ser Ser Gly Asn Gin Cys Val

Thr Gly Glu Gly Thr Pro Lys Pro Gin Ser His Asn Asp Gly Asp Phe Glu Glu H e Pro
Thr Gly Glu Gly Thr Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu H e P I o
Thr Gly Glu Gly Thr Pro Lys Pro Gl n Ser H I S Asn Gin Gly Asp Phe Glu Pro H e Pro
Asp Gly Glu Gly Thr Pro _.y s Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Gl u H e Pro
His Gly Glu Gly Thi Pro Lys Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Glu H e Pro

Glu Glu . Tyr Lru Gin

Glu Glu . Tyr Leu Gin
Glu Asp Ala Tyr Asp Glu
Asp Glu Asp lie l.f'u Asn
Anp Glu A::p 1 l Leu Asn
Fig.3. A comparison of amino acid sequence of some hirudin variants with HMl and HM2. Conserved residues are boxed. Spaces
are introduced tor maximal alignment.

tic peptide 21-Al. RP-HPLC purification of tryptic peptides
from HMl gave two peaks corresponding to the same resi-
dues 3 7 - 4 7 . One of them was completely sequenced and
was shown to have a thrconine residue at position 43. For
the second peak thc presence of thrconine could not be dem-
onstrated by direct N-terminal sequence analysis of the pep-
tide but it was deduced by amino acid analysis as in HM2
(see Fig. 2 and Table 2 in Supplement). These results indi-
rectly suggest the presence of a post-translational modifica-
tion of Thr43.
Each of the three peaks of protein obtained from affinity
chromatography and separated by RP-HPLC was found to be
endowed with antithrombin activity. When assayed with the
fibrin clot test, thc antithrombin activity of homogeneous
purified HM2 (11 (MX) ATU/mg) was found to be approxi-
mately the same as rHVl (11 .^(X) ATU/mg).
Amplirication of RNA saniple.s
Thc existence of highly conserved regions in several hi-
rudin variants and in HMl and HM2 (Fig. 3) was the basis
for the generation of specific primers for PCR amplification
on H. manillensis RNA.
The whole PCR strategy used to isolate HMl and HM2
cDNAs is outlined in Fig. A. For the first round of amplifica-
tion, sense and antisense primers were designed to include
all possible nucleotide combinatiiins that could encode the
conserved regions spanning amino acids 3 - 8 and 5b-52.
The PCR amplification performed on reverse-transcribed
leech head RNA gave a series of bands, where the most
abundant corresponded to the predicted molecular mass. This
band was isolated and subcloned into the plIC vector. Se-
quencing of purified clones yielded the expected HMl and
HM2 cDNA portions.
To obtain 3'-end cDNA sequences we applied thc RACF
protocol |23|. This method allows amplification of regions
of unknown sequence between a specific point in thc tran-
script and the ^ or 3' end. A cDNA-specific primer, spanning
residues 32 — 37. was designed on the basis ofthe previously
determined HM2 sequence (Fig. 4). This was used together
with the dT17 adaptor primer to amplify the reverse-tran-
scribed RNA. A small amount of the PCR mixture was re-
amplified with the same oligonucleotides using more strin-
gent conditions. Two major bands were selected and. after
sequencing, the 3' end of HM2 cDNA was determined.
In order to clone the 5' end of HM1 and HM2 cDNAs
we again followed the RACE protocol. Reverse transcription
of leech RNA was performed using a gene-specific primer
(.•i'RT) designed trom the previously determined C-terminal
nucleotide sequence (see Fig. 4). The first-strand reaction
products were polyadenylated at their 3' ends. Finally. PCR
amplification was accomplished by adding another gene-spe-
cific primer (64-60) upstream to the 5'RT. However,
following electrophoresis on agarose gel. only a broad smear
could be visualized and even a second re-ampliflcation step
carried out in the same conditions did not yield any discrete
band. At this point, thc PCR mixture was used to perform a
Southern blot analysis using as probe the HM2 nucleotide
sequence obtained during the tlrst round of amplification.
Two discrete bands of approximately 3CK) and 360 bp were
finally detected (Fig. 5). These were purified, cloned and se-
quenced leading us to the elucidation of the full-length
cDNAs for the two variants (Fig. 6). The amino acid se-
quences of HMl and HM2. as deduced from the cDNAs. are
identical to the ones obtained by direct chemical sequencing
(results not illustrated).
Amplification of DNA samples and Southern analysis
Little is known of the genomic organization of hirudin or
hirudin-like genes. The availability of cDNA sequences gave
us the opportunity to undertake the isolation of genomic frag-
ments from H. tiumillciisis.
Accordingly, a new primer corresponding to the first
seven amino acids ofthe signal peptide and which could thus
recognize both HMl and HM2 was designed and chemically
300

HM2 VSTTDCTESCQNYCLCVCSNVCCECKNCQLSSSCNQCVHCECTPKPKSQTEGDFEEIPDEDILN

I St round
3-B

2na round
32-37 OHgo dT

3ra round ». .«
ollgoOT 64 60

Fig.4. PCR amplirication strategy u.sed for isolation of HMl and HM2 cDNAs. A rcprcsc-niaiion of HM2 mRNA wilh the corresponding
amino acid sequence, is shown al the top ot ihe figure. Primers used in different rounds of amplification are represented by arrows; the
arrow heads indicate the 3' end of each primer.

B mentioning that the nucleotide sequences of the four exons

of the cloned genes were a confirmation of thc previously
1 2 3 determined cDNA sequences, thus eliminating any doubt on
possible nucleotide misincorporations introduced by the Taq
polymerase.
In order to confirm the presence of introns in these genes,
we performed a .Southern analysis on total DNA extracted
- 1353 from H. manillensi.s head. The genomic DNA preparation
- 1078
- 872
was digested with EcoRX and Ham\\\ whose cutting sites
were not found in thc PCR-amplified genomic fragments. As
- 603 probe, we used thc lull-length cDNA for HMl. As shown in
Fig. 7, even a double digestion with EcoRX and Bani\\\ did
-*- 360 bp not yield any hybridization signal at sizes below 1800 bp,
310
278 -«- 300 bp thus confirming that EcoRX and BamH\ restriction sites, if
234 present in thc HMl or HM2 genes, are indeed external to the
194
genomic fragments obtained by PCR amplification. The large
molecular mass of thc hybridization signals confirmed, in
addition, thc presence of intervening sequences in thc HMl
and HM2 genes.
Fig.5. Kvsult of thf third round of I'CK anipliHcation. (A)
Ethidium-hromide-stained agarose gel containing \5[i\ amplifica-
tion prixlucts (lanes 1 and 2) and the HaelW digest of ft>Xil4 DNA Expression and prrtduction of H1VI2
(lane i) as molecular ma.ss standard. (B) Autoradiograph of the same
gel shown in (A) after hybridization with a 'P-labeled probe. The Fifficient production of recombinant HM2 was achieved
two hybridizing bands of about 36() and 3(K) bp present in lanes 1
using periplasmic expression in /;. coli. according to a strat-
and 2 are marked by an arrow head. egy previously described [27].
Six oligonucleotides, 83-HX) bases long, were prcpiu-cd
hy chemical synthesis and ligated to construct a synthetic
synthesized. As "downslream primer' we selected thc 64 — gene for periplasmic expression of HM2. Thc nucleotide se-
60 primer previously used for the cDNA amplifications. PCR quence was designed on the basis ot preferential codon usage
amplification was performed on total DNA extracted from in E. coli |28|. As shown in Fig. 8 (in .Supplement), thc syn-
the leech head and its products were analysed by agarose gel thetic gene consists of thc oinpA ribosomc-hinding site, fol-
electrophoresis where a single band of approxitnatcly 7(X) bp lowed by the ompA leader peptide and HM2 coding se-
was detected. Following cloning and sequencing, this frag- quences. Thc HM2 gene was then inserted into an expression
ment was found to correspond to the HMl and HM2 genes. plasmid under the control of thc Tip promoter and carrying
As shown in Fig. 6. both gene fragments contain the entire thc tetracycline resistance marker.
coding sequence and indicate thc presence of four exons in- /:. coli strain B, harboring thc HM2 expression plasmid,
terrupted by three introns at the same position. Thc first exon was selected lor thc production of thc protein. The primary
encodes only for the entire signal peptide while thc third translation product of recombinant bacteria consisted in an
exon encodes for the central amino acid sequence which OmpA-lcadcr-pcptide-HM2 fusion protein, which was ex-
shows the lower similarity hetween HMl and HM2. Finally, ported to the periplasmic compartment and correctly pro-
the second and fourth exons encode lor thc N-terminal and cessed, as demonstrated by N- and C-tcrminal analyses.
COOH-terminal domains, respectively. Starting from I 1 bacterial culture, approximately 70 mg
All splice junctions follow the 5'GT/3'AG splice rule HM2 were obtained in thc raw periplasmic extract. A simple
p.**!. Introns, as in many species including plants and Dm- purification protocol with two chromatographic steps gave a
sophila, arc more A + T-rich than Hanking exons |26| and final homogeneous preparation of HM2 with an overall yield
are highly conserved between HMl and HM2. It is worth of 70%.
 .301

n F S L K L F U U F L B U C I C U S g f i U
RTCTTCTCTCTCflnCTICTTCCITCTCnCCTCCCTCTTTCCfiTCTCCCTCTCTCflfiCCnGgtoogoooooettaaltlglctUgtoogoa

S V T 0
ojgaaagtgttgattogoatgtcatgtttotltttgtttgttttggccaatcaaatgocctttttttttttooattcogTGflCCTRCflCTCn

C T E S G O H V C L C U
TTGTRCGGflnTCnCCCCRGnnTTflTTCTCTRTGCGTGgtacgttcgoaatttoctattttttatgctttoaattccccaocoaatgctgtcg
I - t-

C G N L C G G G K N C E n O G S G N K C U O G E
ootcttlogGGRGGTRRTCTCTGCGGTGGRGGCRRRCRTTGTGRRRTGGRCGGTTCTGCftRRTRnOTGCGTCGRTGGGGgtttgltttggca

- S - U - - E - - N - O L S S - - - O - - H - -

otttotgacaoaaattttgotatatttatataoaacaccotagttactcaoagaotgtctgactcooggaoootttattggQgtottttgto
o cc c—get c 0 c

aaccccttttgtttoccgccooaattgotootottgcccgogttocatottoaottgaoogotootggcgoootottottgotcottaotat
.. Q O .........

C T P K P K S O T E G D F E E I P D E D I L N
cotcottttoottgcogRflGGTRCTCCGRRGCCTRRGRGCCRGRCTGRRGGCGRTTTCGRRGRflBTCCCRGflTCRRGflTRTRTTGRRTIfia

Fig.6. Nucleotide and deduced amino acid sequence of HMl and HM2 cDNAs and genomic fragments. Exons are shown in capital
letters; introns are shown in lowercase letters. Ihe deduced amino acids, eticoded by exons. aie shown above the first ba.se ot each ccxlon,
starting with the first ATCJ triplet. Matching nucleotide and atnino acid sequences between the two DNAs are denoted as a dotted Une.
Spaces are introduced to maximize alignment.

ing, peptide mapping and time-course hydrolysis with ear-

1 2 3 bp boxypeptidase. using the methods described above for the
natural molecules.
Besides this, eleetrospray mass spectrometry analy.ses of
-6800 purified HM2 gave an experitnental molecular mass of
6797.25 ±0.63 Da against the value of 6797.23 Da calcu-
-4500 lated for the molecule with the six eysteines all involved in
-4000 disulfide bridges (data not shown).
-3200 The final lyophilized pteparation i>f HM2 displayed high
biochemical activity when its potency as a throtnbin inhibitor
-2100
was assayed. Antithtotnbin activity of reeonibinant HM2 was
5850 Tlil/mg when tneasured by the S-2238 chromogenie
- 1800 test and 11 8(K) ATU/tng. when tneasured with fibrinogen as
substrate. According to these values, thrombin inhibition of
HM2 is fully comparable to the activity of other recotnbinant
hirudin variants [30).
Fig. 7. Southern blot analysis of //. manillensis DNA. Genomie
DNA was diuesteil with EcoRl (lane 1). W(W/1II (latie 2) and EcoRV
BamHl (lane 3). respectively. The blot was hybridized with the ' P-
laheled HMl cDNA. .Sizes of fragments, in bp. were detertiiined by DISCUSSION
reference to the 1-kb molecular mass standard and are indicated at
the right. Most of the polypeptides belonging to the family of hini-
dins and characterized to date ha\e been isolated frotn the
European medicinal leech //. inetlicinalis |9. 10]. This leech
In the purification of HM2 we took advantage of the has been employed in both tnedical practice and pharmaceu-
availability ofa sitigle histiditie residue at positioti 39 atid it.s tical research for many years. Among the possible phamiaco-
ability to complex with iinmohili/cd chclating cations. As logically active substances produced by leeches, hirudin and
expected. HM2 does not bind to ininiobili/ed Zn" ions, bttt its analogs have generated a widespread interest in both in-
it binds well oti Cii ' clielating tesin and ean be eluted in dustrial as well as academic environments due to their strong
good yield by lowering the pH |2y|. On this basis we set up a anticoagulant activity. Indeed, hirudin is the most active and
preparative protocol of HM2 consisting of a cation-exchange specific inhibitor of matntnalian thrombin so far charac-
chtx)tiiatography followed by a Cu' -chelating column. tetizetl.
The complete amino acid sequence of recotnbinant HM2 However, simply on a theoretical basis. H. tiiedicinalis
was found to be ct)nect as verified by N-termitial sequenc- may not be the best choice of leeeh for the isolation of inhibi-
M)2

tors acting against mammalian thrombin. H. medicinatis, in degree of heterogeneity at the genetic level could be present
tact, is primarily an amphibian leech [11] and thus one may in a given set of leeches. Indeed, a possible leech hetero-
speculate that the pool of hirudins produced by such a leech geneity could explain why we were not able to isolate any
is adapted primarily to neutralize amphibian thrombin. clone coding for the hirudin analogs described by Steiner et
Accordingly we focused our attention on a different leech al. 1311.
species. H. manittensis. reported to be primarily a parasite of The cDNA clones coding for the hirudin isotorms HMl
mammals | t t | . In this paper we report the purification, the and HM2 indicate the expression of pre-hirudins of 84 amino
structural characterization and the molecular cloning of two acids where the first 20 residues constitute the signal peptide
novel hirudin-like polypeptides from the Asian leech H. ma- required for extracellular secretion. Interestingly, the HMl
nillensis. We also describe a method to produce a fully active and HM2 signal peptides share a 100% similarity both at the
and correctly processed form of recombinant HM2 in high amino acid and nucleotide level. In addition, from the HV2
yield by expressing in E. coti an OmpA-leader-peptide — cDNA sequence previously reported |13|, it is worth noting
HM2 fusion. that the partial signal peptide is also similar to the corre-
The primary structure of these new polypeptides indi- sponding sequence in HMl and HM2. These data suggest
cates that they share the same general characteristics of the that the secretion signals responsible for the intracellular and
previously known hirudin variants |101. Indeed, an overall extracellular transport of the hirudin isotorms may be highly
707f similarity with HVl and the presence ofthe six cysteine conserved from one leech species to another. Moreover, such
residues in highly conserved positions suggest the presence similarity may offer an extremely valuable technical opportu-
of three disulfide bridges equivalent to those found for HVl. nity to PCR-clone additional isoforms of hirudin from H.
It is. however, interesting to note that most of the vari- manitten.si.s as well as from other leeches by simply using
ability concerns amino acid portions which are not directly as 'upstream primers' oligonucleotides based on the signal
involved in the interaction with thrombin. as shown by the peptide sequence.
resolution of tridjmensional structure of hirudin-thrombin The amino acid divergence between HMl and HM2 is
crystals [7|. All of the hirudin variants extracted from H. limited to the central 'core' region of the hirudin isotorms.
medicinatis and characterized to date contain in the C-termi- From previous structural studies on the interaction of hirudin
nal portion of the molecule a site of sulfation. that is a tyro- with thrombin, it appears that this region is mainly responsi-
sine residue, for example Tyr63 of HVl. The lack of the ble for the structural conformation of the molecule and it is
tyrosine residue in the C-terminal portion of the new hirudin not directly involved in thrombin binding |8|. The genomic
variants from H. manillen.sis is an interesting finding, indicat- organization of HMl and HM2 indicates that the observed
ing that such post-translational modification might have amino acid diversity between the two isoforms is coded by a
evolved differently atnong ditferent sub-families of leeches. precise exon. Interestingly, introns are not ramdomly placed
The fact that we were not able to clone from H. manillensi.s but they correlate with functional elements of the encoded
any hirudin isoform carrying tyrosine residues in the COOH protein.
domain could also imply possible evolutionary ditferences All data reported in this paper demonstrate that these two
between this leech species and H. medieinati.s.
genes are very closely related to each other and support the
While this paper was in preparation. Steiner et al. l.'^l) hypothesis that HMl and HM2 may have evolved by gene
reported the characterization, from a leech purported to be duplication. It will be interesting to elucidate the genomic
H. manillen.si.K, of two other hirudin-like polypeptides which organization of other hirudin isoforms, even trom other
share low similarity with HMl and HM2. One of the de- leeches, to raise considerations on possible evolutionary
scribed sequences also carries a potential sulfation site. A pathways or alternative expression mechanisms.
relevant aspect of the hirudin variants described by Steiner
et al. was the presence of a O-glycosidic chain on a threonine
residue. Combined results from peptide mapping, amino acid Supplement
analysis and sequencing indicate that the corresponding thre- Table 2. Amino acid sequence of tryptic peptides of HMl and
onine residue at position 43 of extracted HMl and HM2 is H1V12. Peptides generated by trypsin digestion were resolved hy RP-
likely to be a putative glycosylation site. Taken together, HPLC (as shown In Fig. 2) and subjected to sequence analysis. Their
these data support the hypothesis that hirudin-like polypep- respective initial yields are reported; X = residue not determined hy
tides from H. manillen.si.s are characteristically modified with amino acid sequencing; * = fragment containing a putative gtyco-
an O-glycosidic chain on threonine. Besides, a family of iso- sylated threonine residue
form variants seems also to be present in H. manitlensi.s. as
is well documented for H. medicinalis. Fragment Sequence Yield
To isolate cDNA and genomic clones from the leech H. pmol
manitten.sis we used the well-established PCR technology
using primers based on the amino acid sequence. In this re- HMt
spect, it is surprising to note that the gene in H. medicinati.s t - 1 .^ VSYTDCTESGQNY .'i65
t4 -26 CLCVGGNLCGGGK 4(K)
coding for the most characterized hirudin isoform, HVl. has 27-36 HCEMDGSGNK 320
never been isolated, while only a partial clone coding for the 37-47 CVDGEGTPKPK 360
isoform HV2 has been rept)rted by Harvey et al. | t 3 | . 37-47(*) CVDGEGXPKPK t63
A quite important feature of our technical approach is the 48-64 SQTEGDFEEIPDEDILN t68
capability of isolating PCR-amplified clones from total RNA HM2
preparations extracted from a very limited number of leeches. t-13 VSYTDCTESGQNY 'M»
In some cases the head of just one specimen was sufficient 14-26 CLCVGSNVCGEGK 654
to yield a quantity of messenger RNA from which we could 27-47 NCQLSSSGNQCVHGEGXPKPK 7t2
amplify and clone the desired cDNA. This is an extremely 48-64 SQTEGDFEEIPDEDILN 220
important aspect since it may not be excluded that a certain
 0.15 r We would like to thank Prof. Piero Pucci (University of Naples)
for electrospray-mass spectrometry analysis of rHM2 and Jan Ma-
lyszko for oligonucleotide synthesis.

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Hindlll oligo

Oa^
oli(o 2

Ball nliKO .1
GT'nX:TTACACCGACT(J:7\CCGAATCIGG 3' i • (XAGAACTACTCCCTCTTGCGTTCgrTCTAACGTTTGCGGTCyAGGTAAAAACTCCCAGCTGTCTT^
CAViGAATGTGGCTX5ACGTGGCTTAGACCGGTCT 5 • 3 • TGATGACGGACACXSiafiCCAAGATTGCAAACGCCACnCCATTTTreAaSSTCGZiCAGAAGA
oligo 4

oligo 5
'IX •VT.TAACCAGl'CCGTTCAC V
Af •Aa-ArTGGTCAa3CAAGTa:CAC 5 • V TTCCATGGaXn-TTGCCirrAGAGTCTCACTIXXACTGAAGCTIXrrnAAGGCC^^
oligo 6

Ramlll
TAi n'AAtJ f

Fig.8. Nucleotide sequence of the six oligonucleotides coding for HM2. The portion coding for the OmpA leader peptide is shown m
bold face. The restriction sites HinttUl and Bam[U were introduced to facilitate cloning.
304
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