0049-3848/78/0601-1135
THROMBOSIS RESEARCH Vol. 12, pp. 1135-I 146. 8 Pcrgamon Press Ltd. 1978. Printed in Great Britain.
u32.oOiO
THE AM140 AC13 SEQUENCE OF THE CARBOXY-TERMINAL 142 AMINO ACIDS OF THE s-CHAIN OF HUMAN FIBRINOGEN Barbara A. Cottrell anu Russell F. Doolittle Department of Chemistry University of California, San Diego La Jolla, California 92093 U.S.A.
(Received
ABSTRACT
15.4.1978.
Accepted
by
Editor
E. Xihalyi)
The amino acid sequence of the carboxy-terminal 142 residues of the u-chain of human fibrinogen has been determined. The sequence was established by determining the structures of cyanogen bromide fragments and key overlap peptides isolated from enzymatic digests of a-chains. One of the cyanogen bromide fragments is a peptide we have previously shown to participate in the a-chain cross-linking system.
INTRODUCTION Almost the entire amino acid sequence of the amino-terminal half of the human fibrinogen a-chain--amounting to 272 residues--has been assembled on the basis of reports from several different laboratories (l-6).
Moreover,
we have reported a characterization of the eleven cyanogen bromide fragments which comprise a-chains and proposed an arrangement for all but one of them. We now report the amino acid sequence of the carboxy-terminal 142 residues of the a-chain, including the previously unplaced fragment.
MATERIALS AND METHODS Most of the materials and procedures used in this study have been fully described in previous publications from this laboratory.
In particular, we
have already provided the details for the isolation of the eleven constituent 1135
1136
h-CHAIY
OF HTXAN
FIBRINOGEN
cyanogen bromide fragments of human fibrinogen z-chains (7).
Sol.l2,No.6
The sequences
of these fragments have been determined by a variety of procedures, but especially by a solid phase thioacetylation stepwise degradation procedure (8), both in manual and automated modes (9); the approach has been supplemented by the dimethylaminonaphthylsulfonyl chloride-phenylisothiocyanate (DNSPhNCS) procedure (10).
Because the yield of serine and threonine are poor
with the thioacetylation method, all serines and threonines have been independently determined by the ONS-PhNCS procedure.
Subpeptides of the cyano-
gen bromide fragments were generated by several enzymes, including trypsin (Worthington), chymotrypsin (Worthington), staphylococcal protease X (Miles) and thermolysin (Daiwa Kasei KK, Osaka).
In addition, some key overlap
fragments were obtained by plasmin (Kabi, Stockholm) digestionof fibrinogen and the staphylococcal protease digestion of entire a-chains.
Peptides were
purified by gel filtration on appropriate Sephadex columns and by paper electrophoresis at pH 6.5 and/or pH 2.0.
Amino acid compositions were deter-
mined after total acid hydrolysis, using either a Spinco Model 121M or Spinco >lodel 119 automatic amino acid analyzer.
Amides were established either on
the basis of peptide mobilities at pH 6.5 (11) or by susceptibility to staphylococcal protease (12).
RESULTS Fragment CN- IT1 This fragment is actually the carboxy-terminal tryptic peptide of the largest cyanogen bromide fragment (CM-I) obtained from the a-chains. 15 10 5 Glu-Val-Val-Thr-Ser-Glu-Asp-Gly-Ser-Asp-Cys-Pro-Glu-Ala-Gln-Met
--+
--w-t
-
-4+--+
--+--++4
I’ Gl
Fig. 1.
Data used to obtain the amino acid sequence of CN-ITl. The subpeptides obtained with staphylococcal protease begin with the designation G. Full arrows (-) represent successful identification by solid phase thioacetylation. Half arrows (-_) represent successful determinations by DWS-PhNCS.
It was
.c. h
m
--h---h---
7cu~cu*-~~cu~ ----vu--vu
+ 5--3~cu0,0ru0%cn . . . . . . . c-4
~cvml-~s-~o
.
. ,
I
00~07 . UN--
.
.
.
ti-CHXIS
1133
OF
Kc?lA?j
Vo1.12,No.6
FIBRI?iOGES
readily isolated from a tryptic digest o-f C:+-i (each digest 30 mg, ca 1 umole) because of the cysteine residue which had been labelled with 14-C iodoacetic acid during the original reduction and alkylation of the fibrinogen prior to chain separation.
The peptide contains homoserine but no argi-
nine or lysine and is composed of 16 amino acids (Table I).
It was attached
to glass beads by the homoserine method (13) and subjected to degradation by the thioacetylation procedure.
Successful identifications were made through
residue 13, although the serine and threonine identifications were equivocal. The
cysteine
at residue
11 was easily identified by the radioactivity which
was released at that step.
The constituent subpeptides were obtained by
digestion of a-chains with the staphylococcal protease (12), and these sequenced by the DRS-PhNCS procedure (Fig.
1).
Fragment CN-VIE In our previous report on the cyanogen bromide fragments of a-chains (7) we noted that we had been unable to purify this fragment (CN-VIB), but that we had been able to make a preliminary characterization on the basis of two tryptic peptides which were purified from digests of other fragments.
We
have now been successful in purifying the fragmelitby paper electrophoresis
---t++ I-
10 5 15 20 Asp-Leu-Gly-Thr-Leu-Ser-Gly-Ile-Gly-Thr-Leu-Asp-Gly-Phe-Arg-His-Arg-His-Pro-Asp-c-t
-
--
---c-c
-+++~~--w~~~~
Tl --7-7-v--7---7 -CHl-
Tb
-MH4b
BH3---
c---CH4-
40 25 30 35 Glu-Ala-Ala-Phe-Phe-Asp-Thr-Ala-Ser-Thr-Gly-Lys-Thr-Phe-Pro-Gly-Phe-Phe-Ser-Pro-Met
Fig. 2.
Summary of data used in determining CH = chymotrypsin;
T = trypsin.
the sequence of fragment CN-VIS. Other designations as in Fig. 1.
2t pH 5.5, however, and its amino acid composition is presented in Table I. The fragment contains 41 residues and yields three subpeptides upondigestion with trypsin.
The complete sequence was determined by a series of thio-
acetylation runs, first on the intact fragment, and then on the middle and carsoxqr-terminal tryptic peptides (Fig. 2).
In addition, two of the tryp-
tic peptides were further fragmented with chymotrypsin, and verification of the sequences determined by the DNS-PhtKS method.
Fragment WIVB This fragment contains 59 residues, the sequence of more than half of which we reported on a previous occasion (7).
The fragment is of considera-
ble interest because it constitutes a portion of the z-chain cross-linking system (14).
The completion of the sequence was accomplished by a variety
of techniques, the major features of which are sullxnarized in Fig. 3.
It
should be mentioned that an unexpected cleavage occurs with staphylococcal protease, the lysyl-glutamine bond at position 40-41 being broken (Fig. 3). As a result, no overlap has been obtained for this region, and further studies to confirm this section are planned.
5 10 15 20 25 30 Ltu-Gly-Glu-Phe-V~1-Ser-Glu-Thr-Giu-Ser-Arg-Gly-Ser-Glu-Ser-G~y-~le-Phe-Thr-Asn-Thr-Lys-Glu-Ser-S~r-Ser-~l~-Hi~-Pr~G~y
_____
__
__
__-..
__
___-
--
-
-_
-
-
---..-.-
35 45 55 40 50 Ile-Ala-Glu-Phe-Pro-Ser-Arg-Gly-Lyr-Gln-Ph~~lhr;S~r,S~r~lyr-Asn-A~g-Gly-As~-S~r-Thr-Phe-Glu-Ser-Lys-S~r-lyr-Lys-~t
Fig. 3.
Summary of data used to determine the amino acid sequence of fragment C&IVB. See legends of Figs. 1 and 2 for explanation of symbols.
a,-CH.XIX
1140
-Fr-rent
OF
HI_-VXS
Vol.l2,No.6
FIBRIXOGES
Ch-VIC
Fragment CN-VIC is the carboxy-terminal cyanogen bromide fragment of I-cii~ins. AS such it tioes not contain homoserine. reported the amino acid sequence of this X-residue
We have previously long peptide (15), but
a summary of the data is provided in Fig. 4.
5 10 15 20 Ala-Asp-Glu-Ala-Gly-Ser-Glu-Ala-Asp-His-Glu-Gly-Thr-His-Ser-Thr-Lys-Arg-Gly-His-Aia-Lys-Ser-Arg-Pro-Val
25
_---.4-..-______-_
Tl
IT&
_'41,-
-"--/
_)--77--7--r-Y-77 -Th3II
Fig. 4.
Data used to establish sequence of fragment CN-VIC. T = trypsin; Th = thermolysin. Other designations as in Fig. 1. The peptide was also fragmented with staphylococcal protease (data not shown).
Overlap Peptides The overlap peptides which span the four cyanogen bromide fragments involved in the carboxy-terminal segment of a-chains were obtained in two different ways.
First, the overlap between CN-I and CN-VI6 was isolated
from a staphylococcal protease digestion of a-chains. Ala-Gln-Met-Asp.
It
had the sequence
The peptide corresponding to the junction of CN-VIB and
CM-IVB was isolated from a two hr. plasmic digest of fibrinogen.
A methio-
nine-containing and threonine-ending fragment was obtained and further digested with trypsin; the 20-residue amino-terminal peptide was purified and characterized.
Its composition corresponded to the observed sequences
at the junction of CSVIB
and CN-IVB (Fig. 5).
The junction between CN-IVB and CN-VIC, although the only possibility remaining by default, was confirmed by the plasmic cleavage of a 27-residue methionine ending peptide which in all other respects is identical with the X-residue
carboxy-terminal cyanogen bromide fragment (15).
VOL.12
d-CHAI?i
so.0
OF
HWXB
1141
FTBRISOGET
Thr-Le~-Asp-Gly-Phe-Arg-His-Arg-His-Pro-Asp-Glu-A1a-A1a-Phe-Phe-Asp-Thr-Ala-Ser-Thr-G1y-Lys-Thr-Phe30 40
50
Pro-G1y-Phe-Phe-Ser-Pr~~t-Leu-Gly-Glu-Phe-V~l-Ser-G~u-Thr-G1u-Ser-Arg-G1y-Ser-G1u-Ser-G1y-I1e-Phe60 70
Thr-Asn-Thr-Lys-G1u-Ser-Ser-Ser-His-His-Pro-G1y-Ile-Ala-Glu-Phe-Pro-Ser-Arg-G1y-Lys-Gln-Phe~Thr~Ser, 60 go
100
Ser~Tyr-Asn-Arg-Gly-Asp-Ser-iht-Phe-Glu-Ser-Lyr-Ser-Tyr-Lys~~et-Ala-Asp-Glu-~~~-Gty-Ser-Glu-Ala-Asp110
Hls-Glu-Gly-Thr-His-Ser-Thr-Lys-Arg-Gly-His-Al~-Lys-Ser-Arg-Pro-Val 130 140
Fig. 5.
Carboxy-terminal 142 residues of a-chain from human fibrinogen. Residue numbers 1-142 correspond approximately to residues 485-626 in the a-chain. Known plasmin cleavage points are denoted with arrows (4).
DISCUSSION
The structure of the carboxy-terminal half of the fibrinogen a-chain is of interest on several counts.
First, it is well known that these portions
of the fibrinogen molecule are highly exposed and readily removed and/or degraded by a variety of proteases.
Moreover, the amino acid composition
of these exposed portions has been reported to be very polar and deficient in nonpolar amino acids (3, 4).
In this article we report the amino acid
sequence of the carboxy-terminal 142 residues, a region accounting for not quite one-quarter of the u-chain of human fibrinogen. The noteworthy features of the sequence (Fig. 5) can be categorized as follows:
(a)
nature of the amino acids involved, (b) degree of homology
with 5- and y-chains and internal homologies, (c)bonds vulnerable to plasmic cleavage and potential sites for other proteases, and (d) localization of a-chain cross-linking sites. Amino Acid Composition While it must be borne in mind that 142 residues is a relatively small number of amino acids about which to generalize, there are several points which ought to be mentioned here, if not dwelt upon.
First, althoilgh the
composition of the last quarter of the a-chain is generally polar, it is not excessively so, especially when compared with the adjacent (middle, or third
1142
d-CHAIX
quarter) segment.
OF HUMAX
FIBRIZ;OGEN
Vo1.12.No.6
Fully 30% of the residues are nonpolar (Ala, Pro, Cys,
Val, Met, Ile, Leu, Tyr, or Phe).
There are no tryptophan residues,however,
and only two tyrosines, a practical point which meant that the fragr,lents have little or no absorbance at 280 nm. phenylalanines, however.
Eleven of the 142 residues are
The most predominant amino acid is serine (21 of
lS2 residues), and serine and threonine together account for 20% (34 of 142) of the sidechains.
Negatively charged residues (glutamic and aspartic
acids) outnumber positively charged ones (arginines and lysines) by almost two to one (24 to 14).
The proline content is an average 4.9% (7 of 142).
Secondary Structure Prediction A preliminary analysis of the sequence of the C-terminal 126 residues was kindly undertaken by Dr. Jean Garnier using a still unpublished procedure (Garnier, J., Osguthorpe, D. J. and Robson, B., Unpublished Procedure). The analysis indicates that most of this region is consistent with a random coil or other undefined structure which may include a number of reverse turns.
The only sections with any significant probability of a-helix were
residues 33-43 and 114-133 in Fig. 5 (approximately 517-527 and 598-617 in the intact a-chain).
As noted in an earlier report from this laboratory (15), the homology at the carboxy-terminus between w
and y-chains is marginal at best, in
contrast to the obvious homology between B- and y-chains (16).
This trend
has been borne out by the additional sequence information, there being no obvious relationship between the carboxy-terminal quarter of a-chains and the corresponding sequences in B- and y-chains, which themselves exhibit very strong homologies (17, 18).
On the other hand, there is a hint of
internal homology in this part of the u-chain, and we would cautiously suggest that the a-chain carboxy-terminal segment has been elongated by a series of contiguous gene duplications, 50 to 60 amino acids in length (Fig. 6).
It is now well established that the three constituent chains of
of fibrinogen, Q-, 3- and Y-, are homologous at their amino-terminal ends (19).
The cc-chain,which is the most variable of the three chains on
a molecular weight basis(20).,appears to have been less restricted in its evolution at the carboxy-terminus.
d_CHdIN
x-01.12.So.h
1143
OF Hl_?l;tS FIBRISOGES
Phe-Asp-Thr-Ala Pro-
Fig. 6.
.
.
.
Possible internal homologies in the carboxy-terminal portion of the u-chain of human fibrinogen. The data suggest that a series of contiguous duplications 50-60 residues long may have led to elongation of the a-chain cat-boxy-terminal region. Numbering based on Fig. 5.
Plasmin Digestion Points These studies have led
to the identification of two plasmin attack
points near the carboxy-terminal of the a-chains. ages occurs at theLys-Metbond
The first of these cleav-
which corresponds approximately to residues
600-601 in the a-chain (115-116 in Fig. 5).
As we have reported previously
(15),this is apparently the very first plasmin attack point; it also occurs in vivo and leads to separable aichains and aZchains.
A second early plas-
min cleavage occurs at the Lys-Thr bond denoted at residues 48-49 in Fig. 5 (approximately residues 567-568 in the u-chain). Arginyl-Glycine Bonds There are four arginyl-9lycine peptide bonds in this quarter of the a-chain, about ten times more than would be expected on a strictly random basis.
As is well known, thrombin attacks only arginyl-glycine bonds when
releas ing fibrinopeptides, regardless of species. narrow specificity when activating factor XIII
(2!),
It also adheres to this and it will be inter-
esting to find if any of these bonds are cleaved during the thrombincataly zed fibrinolysis which has been reported recently (22).
llttlr
OF HWU?i Of.-CHXI1L‘
Vol.l2,No.6
FIBRIBOGEN
a-Chain Cross-Linking Sites The carboxy-terminal segments of a-chains also contain a portion of the z-chain
multimeric cross-linking system.
We have previously shown that
cyanogen bromide digestion of fully cross-linked fibrin generates a crosslinked polymer comprised of two fragments, one of which (C&I)
stretches
approximately from residues 241-500, and the other of which (CN-IVB)
is
the
penultimate cyanogen bromide fragment in the chain and corresponds approximately to residues 558-607 (14). and only one glutamine.
The latter fragment contains four lysines
Studies are under way to find which of these are
involved in the cross-linking. Status of the Remainder of the a-Chain Sequence At this point approximately two-thirds of the a-chain amino acid sequence has been reported, including the first 272 residues from the aminoterminus (23) and the 142 residues at the carboxy-terminus.
The 210 :!lO
residues which comprise the intervening region all occur in a single cyanogen bromide fragment (CN-I);
the region is distinctive in that it contains
a very large number of serine and glycine residues.
So far we have isolated
and characterized 22 tryptic peptides from this region, and an effort to obtain suitable overlaps by alternative enzymatic digestion is currently under way in order to complete the sequence.
ACKNOWLEDGEMENTS We would like to thank Marcia Riley for preparing fibrinogen and achains,and Dennis Trovato for the operation of amino acid analyzers.
This
study has been supported by USPHS Grants do. HL-18,576 and GM-17,702. REFERENCES 1.
ItiAiiAGA, S., WALLEN, P., GRG:iDAHL, 1'4. J., HENSCHEN, A. AND BLOMBACK, B. On the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digest. Eur.J. Biochem. 8, 189, 169.
2.
TAKAGI, T. and DOOLITTLE, R. F. The amino acid sequences of those portions of human fibrinogen fragment E which are not included inthe anincterminal disulfide knot. Thromb. Res. 7, 813, 1975.
3.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence studies on the achain of human fibrinogen. Location of four plasmin attack points and a covalent cross-linking site. Biochemistry. 14, 5149, 1975.
1145
4.
HARFEttIST, E. J. and CANFIELD, R. E. Degradat ion of fibrinogen by plasi!lirl. IS0lation of an early cleavage product. Biochemistrv. 14, 4110, 1975.
5.
DOOLITTLE, R. F., CASSMAN, K. G., COTTRELL, B. A ., FRIEZNER, 5. TAKAGI, T. Amino acid sequence studies on the a-chain of human gen. Covalent structure of the 1-chain portion of fragment D. 16, 1710, 1977. chemistry.
6.
GARDLUND, B. Human fibrinogen--amino acid sequence of fragment E and of adjacent structure in the Aa- and Es-chains. Thromb. Res. 10, 689, 1977.
7.
DOOLITTLE, R. F., CASSMAN, K. G., COTTRELL, B. A., FRIEZNER, S. J - , HUCKO, J. T. and TAKAGI, T. Amino acid sequence studies on the c(chain of human fibrinogen, Characterization of 11 cyanogen bromide fragments. Biochemistry. 16, 1703, 1977.
J. and fibrinoBio-
MROSS, G. A. and DOOLITTLE, R. F. Step-wise degradation of peptides attached to solid supports. Mol. Biol., Biochem. & Biophys. 25, 1, 1977. 9.
DOOLITTLE, L. R., MROSS, G. A., FOTHERGILL, L. A. and DOOLITTLE, R. F. A simple solid-phase amino acid sequencer employing a thioacetylation stepwise degradation procedure. Anal. Biochem. 78, 491, 1977.
10.
GRAY, W. Sequence analysis with dansyl chloride. 333, 1972.
11.
OFFERD, R. E. Electrophoretic mobilities of peptides on paper and their use in the determination of amide groups. Nature (London). 211, 591, 1966.
12.
HOUMARD, J. and DRAPEAU, G. R. Staphylococcal protease: a proteolytic enzyme specific for glutamyl bonds. Proc. Natl. Acad. Sci., U. S. 69, 3506, 1972
13.
HORN, M. J. and LAURSE:i, R. .A. Solid phase Edman degradation: attachment of carboxyl-terminal homoserine peptides to an insoluble resin. FEBS Letters. 36, 285, 1973.
14.
DOOLITTLE, R. F. , CASSMAN, K. G., COTTRELL, B. A. AND FRIEZNER, S. J, Amino acid sequence studies on the a-chain of human fibrinogen. Isolation and characterization of two linked a-chain cyanogen bromide fragments from fully cross-linked fibrin. Biochemistry. 16, 1715, 1977.
15.
COTTRELL, B. A. and DOOLITTLE, R. F. The amino acid sequence of a 27-residue peptide released from the a-chain carboxy-terminus during the plasmic digestion of human fibrinogen. Biochem. Biophys. Res. Comm. 71, 754, 1977.
16.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence of the carboxyterminal cyanogen bromide peptide of the human fibrinogen s-chain: Homology with the corresponding y-chain peptide and presence in fragment D. Biochim. Biophys. Acta. 386 , 617, 1975.
17.
HENSCHEN, A. and LOTTSPEICH, R. Sequence homology between a-chain and s-chain in human fibrin. Thromb. Res. 1 , 869, 1977.
18.
WATT, K. W. K , TAKAGI, T. and DOOLITTLE, R. F. The amino acid sequence of the a-chain of human fibrinogen: homo ogy with the y-chain. Proc. iIat1.Acad. Sci., U.S. In Press.
Meth. Enzymol. 25,
1146
ti -CHAI?i OF
HGXA?r FIBRINOGES
Vo1.12,~0.6
19.
DOOLITTLE, R. F. The evolution of vertebrate fibrinogen. Proceedings. 35, 2145, 1976.
Federation
20.
DOOLITTLE, R. F. Structural aspects of the fibrinogen to fibrin conversion. Adv. Prot. Chem. 27, 1, 1973.
21.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. Biochemistry. 13, 750, 1974.
22.
KANG, E. P. and TRIANTAPHYLLOPOULAS, D. C. Fibrin digestion with thrombin. Comparison with plasmin-digested fibrinogen. Biochim. Biophys. Acta. 490, 430, 1977.
23.
DOOLITTLE, R. F., WATT, K. W. K., COTTRELL, B. A. and TAKAGI, T. Fibrino. gen: A highly evolved regulator agent for maintaining the integrity of the vertebrate circulatory system. Intern. Symp. Proteins, Taipei, 1973. Academic Press. In Press.
THROMBOSIS RESEARCH Vol. 12, pp. 1135-I 146. 8 Pcrgamon Press Ltd. 1978. Printed in Great Britain.
u32.oOiO
THE AM140 AC13 SEQUENCE OF THE CARBOXY-TERMINAL 142 AMINO ACIDS OF THE s-CHAIN OF HUMAN FIBRINOGEN Barbara A. Cottrell anu Russell F. Doolittle Department of Chemistry University of California, San Diego La Jolla, California 92093 U.S.A.
(Received
ABSTRACT
15.4.1978.
Accepted
by
Editor
E. Xihalyi)
The amino acid sequence of the carboxy-terminal 142 residues of the u-chain of human fibrinogen has been determined. The sequence was established by determining the structures of cyanogen bromide fragments and key overlap peptides isolated from enzymatic digests of a-chains. One of the cyanogen bromide fragments is a peptide we have previously shown to participate in the a-chain cross-linking system.
INTRODUCTION Almost the entire amino acid sequence of the amino-terminal half of the human fibrinogen a-chain--amounting to 272 residues--has been assembled on the basis of reports from several different laboratories (l-6).
Moreover,
we have reported a characterization of the eleven cyanogen bromide fragments which comprise a-chains and proposed an arrangement for all but one of them. We now report the amino acid sequence of the carboxy-terminal 142 residues of the a-chain, including the previously unplaced fragment.
MATERIALS AND METHODS Most of the materials and procedures used in this study have been fully described in previous publications from this laboratory.
In particular, we
have already provided the details for the isolation of the eleven constituent 1135
1136
h-CHAIY
OF HTXAN
FIBRINOGEN
cyanogen bromide fragments of human fibrinogen z-chains (7).
Sol.l2,No.6
The sequences
of these fragments have been determined by a variety of procedures, but especially by a solid phase thioacetylation stepwise degradation procedure (8), both in manual and automated modes (9); the approach has been supplemented by the dimethylaminonaphthylsulfonyl chloride-phenylisothiocyanate (DNSPhNCS) procedure (10).
Because the yield of serine and threonine are poor
with the thioacetylation method, all serines and threonines have been independently determined by the ONS-PhNCS procedure.
Subpeptides of the cyano-
gen bromide fragments were generated by several enzymes, including trypsin (Worthington), chymotrypsin (Worthington), staphylococcal protease X (Miles) and thermolysin (Daiwa Kasei KK, Osaka).
In addition, some key overlap
fragments were obtained by plasmin (Kabi, Stockholm) digestionof fibrinogen and the staphylococcal protease digestion of entire a-chains.
Peptides were
purified by gel filtration on appropriate Sephadex columns and by paper electrophoresis at pH 6.5 and/or pH 2.0.
Amino acid compositions were deter-
mined after total acid hydrolysis, using either a Spinco Model 121M or Spinco >lodel 119 automatic amino acid analyzer.
Amides were established either on
the basis of peptide mobilities at pH 6.5 (11) or by susceptibility to staphylococcal protease (12).
RESULTS Fragment CN- IT1 This fragment is actually the carboxy-terminal tryptic peptide of the largest cyanogen bromide fragment (CM-I) obtained from the a-chains. 15 10 5 Glu-Val-Val-Thr-Ser-Glu-Asp-Gly-Ser-Asp-Cys-Pro-Glu-Ala-Gln-Met
--+
--w-t
-
-4+--+
--+--++4
I’ Gl
Fig. 1.
Data used to obtain the amino acid sequence of CN-ITl. The subpeptides obtained with staphylococcal protease begin with the designation G. Full arrows (-) represent successful identification by solid phase thioacetylation. Half arrows (-_) represent successful determinations by DWS-PhNCS.
It was
.c. h
m
--h---h---
7cu~cu*-~~cu~ ----vu--vu
+ 5--3~cu0,0ru0%cn . . . . . . . c-4
~cvml-~s-~o
.
. ,
I
00~07 . UN--
.
.
.
ti-CHXIS
1133
OF
Kc?lA?j
Vo1.12,No.6
FIBRI?iOGES
readily isolated from a tryptic digest o-f C:+-i (each digest 30 mg, ca 1 umole) because of the cysteine residue which had been labelled with 14-C iodoacetic acid during the original reduction and alkylation of the fibrinogen prior to chain separation.
The peptide contains homoserine but no argi-
nine or lysine and is composed of 16 amino acids (Table I).
It was attached
to glass beads by the homoserine method (13) and subjected to degradation by the thioacetylation procedure.
Successful identifications were made through
residue 13, although the serine and threonine identifications were equivocal. The
cysteine
at residue
11 was easily identified by the radioactivity which
was released at that step.
The constituent subpeptides were obtained by
digestion of a-chains with the staphylococcal protease (12), and these sequenced by the DRS-PhNCS procedure (Fig.
1).
Fragment CN-VIE In our previous report on the cyanogen bromide fragments of a-chains (7) we noted that we had been unable to purify this fragment (CN-VIB), but that we had been able to make a preliminary characterization on the basis of two tryptic peptides which were purified from digests of other fragments.
We
have now been successful in purifying the fragmelitby paper electrophoresis
---t++ I-
10 5 15 20 Asp-Leu-Gly-Thr-Leu-Ser-Gly-Ile-Gly-Thr-Leu-Asp-Gly-Phe-Arg-His-Arg-His-Pro-Asp-c-t
-
--
---c-c
-+++~~--w~~~~
Tl --7-7-v--7---7 -CHl-
Tb
-MH4b
BH3---
c---CH4-
40 25 30 35 Glu-Ala-Ala-Phe-Phe-Asp-Thr-Ala-Ser-Thr-Gly-Lys-Thr-Phe-Pro-Gly-Phe-Phe-Ser-Pro-Met
Fig. 2.
Summary of data used in determining CH = chymotrypsin;
T = trypsin.
the sequence of fragment CN-VIS. Other designations as in Fig. 1.
2t pH 5.5, however, and its amino acid composition is presented in Table I. The fragment contains 41 residues and yields three subpeptides upondigestion with trypsin.
The complete sequence was determined by a series of thio-
acetylation runs, first on the intact fragment, and then on the middle and carsoxqr-terminal tryptic peptides (Fig. 2).
In addition, two of the tryp-
tic peptides were further fragmented with chymotrypsin, and verification of the sequences determined by the DNS-PhtKS method.
Fragment WIVB This fragment contains 59 residues, the sequence of more than half of which we reported on a previous occasion (7).
The fragment is of considera-
ble interest because it constitutes a portion of the z-chain cross-linking system (14).
The completion of the sequence was accomplished by a variety
of techniques, the major features of which are sullxnarized in Fig. 3.
It
should be mentioned that an unexpected cleavage occurs with staphylococcal protease, the lysyl-glutamine bond at position 40-41 being broken (Fig. 3). As a result, no overlap has been obtained for this region, and further studies to confirm this section are planned.
5 10 15 20 25 30 Ltu-Gly-Glu-Phe-V~1-Ser-Glu-Thr-Giu-Ser-Arg-Gly-Ser-Glu-Ser-G~y-~le-Phe-Thr-Asn-Thr-Lys-Glu-Ser-S~r-Ser-~l~-Hi~-Pr~G~y
_____
__
__
__-..
__
___-
--
-
-_
-
-
---..-.-
35 45 55 40 50 Ile-Ala-Glu-Phe-Pro-Ser-Arg-Gly-Lyr-Gln-Ph~~lhr;S~r,S~r~lyr-Asn-A~g-Gly-As~-S~r-Thr-Phe-Glu-Ser-Lys-S~r-lyr-Lys-~t
Fig. 3.
Summary of data used to determine the amino acid sequence of fragment C&IVB. See legends of Figs. 1 and 2 for explanation of symbols.
a,-CH.XIX
1140
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HI_-VXS
Vol.l2,No.6
FIBRIXOGES
Ch-VIC
Fragment CN-VIC is the carboxy-terminal cyanogen bromide fragment of I-cii~ins. AS such it tioes not contain homoserine. reported the amino acid sequence of this X-residue
We have previously long peptide (15), but
a summary of the data is provided in Fig. 4.
5 10 15 20 Ala-Asp-Glu-Ala-Gly-Ser-Glu-Ala-Asp-His-Glu-Gly-Thr-His-Ser-Thr-Lys-Arg-Gly-His-Aia-Lys-Ser-Arg-Pro-Val
25
_---.4-..-______-_
Tl
IT&
_'41,-
-"--/
_)--77--7--r-Y-77 -Th3II
Fig. 4.
Data used to establish sequence of fragment CN-VIC. T = trypsin; Th = thermolysin. Other designations as in Fig. 1. The peptide was also fragmented with staphylococcal protease (data not shown).
Overlap Peptides The overlap peptides which span the four cyanogen bromide fragments involved in the carboxy-terminal segment of a-chains were obtained in two different ways.
First, the overlap between CN-I and CN-VI6 was isolated
from a staphylococcal protease digestion of a-chains. Ala-Gln-Met-Asp.
It
had the sequence
The peptide corresponding to the junction of CN-VIB and
CM-IVB was isolated from a two hr. plasmic digest of fibrinogen.
A methio-
nine-containing and threonine-ending fragment was obtained and further digested with trypsin; the 20-residue amino-terminal peptide was purified and characterized.
Its composition corresponded to the observed sequences
at the junction of CSVIB
and CN-IVB (Fig. 5).
The junction between CN-IVB and CN-VIC, although the only possibility remaining by default, was confirmed by the plasmic cleavage of a 27-residue methionine ending peptide which in all other respects is identical with the X-residue
carboxy-terminal cyanogen bromide fragment (15).
VOL.12
d-CHAI?i
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OF
HWXB
1141
FTBRISOGET
Thr-Le~-Asp-Gly-Phe-Arg-His-Arg-His-Pro-Asp-Glu-A1a-A1a-Phe-Phe-Asp-Thr-Ala-Ser-Thr-G1y-Lys-Thr-Phe30 40
50
Pro-G1y-Phe-Phe-Ser-Pr~~t-Leu-Gly-Glu-Phe-V~l-Ser-G~u-Thr-G1u-Ser-Arg-G1y-Ser-G1u-Ser-G1y-I1e-Phe60 70
Thr-Asn-Thr-Lys-G1u-Ser-Ser-Ser-His-His-Pro-G1y-Ile-Ala-Glu-Phe-Pro-Ser-Arg-G1y-Lys-Gln-Phe~Thr~Ser, 60 go
100
Ser~Tyr-Asn-Arg-Gly-Asp-Ser-iht-Phe-Glu-Ser-Lyr-Ser-Tyr-Lys~~et-Ala-Asp-Glu-~~~-Gty-Ser-Glu-Ala-Asp110
Hls-Glu-Gly-Thr-His-Ser-Thr-Lys-Arg-Gly-His-Al~-Lys-Ser-Arg-Pro-Val 130 140
Fig. 5.
Carboxy-terminal 142 residues of a-chain from human fibrinogen. Residue numbers 1-142 correspond approximately to residues 485-626 in the a-chain. Known plasmin cleavage points are denoted with arrows (4).
DISCUSSION
The structure of the carboxy-terminal half of the fibrinogen a-chain is of interest on several counts.
First, it is well known that these portions
of the fibrinogen molecule are highly exposed and readily removed and/or degraded by a variety of proteases.
Moreover, the amino acid composition
of these exposed portions has been reported to be very polar and deficient in nonpolar amino acids (3, 4).
In this article we report the amino acid
sequence of the carboxy-terminal 142 residues, a region accounting for not quite one-quarter of the u-chain of human fibrinogen. The noteworthy features of the sequence (Fig. 5) can be categorized as follows:
(a)
nature of the amino acids involved, (b) degree of homology
with 5- and y-chains and internal homologies, (c)bonds vulnerable to plasmic cleavage and potential sites for other proteases, and (d) localization of a-chain cross-linking sites. Amino Acid Composition While it must be borne in mind that 142 residues is a relatively small number of amino acids about which to generalize, there are several points which ought to be mentioned here, if not dwelt upon.
First, althoilgh the
composition of the last quarter of the a-chain is generally polar, it is not excessively so, especially when compared with the adjacent (middle, or third
1142
d-CHAIX
quarter) segment.
OF HUMAX
FIBRIZ;OGEN
Vo1.12.No.6
Fully 30% of the residues are nonpolar (Ala, Pro, Cys,
Val, Met, Ile, Leu, Tyr, or Phe).
There are no tryptophan residues,however,
and only two tyrosines, a practical point which meant that the fragr,lents have little or no absorbance at 280 nm. phenylalanines, however.
Eleven of the 142 residues are
The most predominant amino acid is serine (21 of
lS2 residues), and serine and threonine together account for 20% (34 of 142) of the sidechains.
Negatively charged residues (glutamic and aspartic
acids) outnumber positively charged ones (arginines and lysines) by almost two to one (24 to 14).
The proline content is an average 4.9% (7 of 142).
Secondary Structure Prediction A preliminary analysis of the sequence of the C-terminal 126 residues was kindly undertaken by Dr. Jean Garnier using a still unpublished procedure (Garnier, J., Osguthorpe, D. J. and Robson, B., Unpublished Procedure). The analysis indicates that most of this region is consistent with a random coil or other undefined structure which may include a number of reverse turns.
The only sections with any significant probability of a-helix were
residues 33-43 and 114-133 in Fig. 5 (approximately 517-527 and 598-617 in the intact a-chain).
As noted in an earlier report from this laboratory (15), the homology at the carboxy-terminus between w
and y-chains is marginal at best, in
contrast to the obvious homology between B- and y-chains (16).
This trend
has been borne out by the additional sequence information, there being no obvious relationship between the carboxy-terminal quarter of a-chains and the corresponding sequences in B- and y-chains, which themselves exhibit very strong homologies (17, 18).
On the other hand, there is a hint of
internal homology in this part of the u-chain, and we would cautiously suggest that the a-chain carboxy-terminal segment has been elongated by a series of contiguous gene duplications, 50 to 60 amino acids in length (Fig. 6).
It is now well established that the three constituent chains of
of fibrinogen, Q-, 3- and Y-, are homologous at their amino-terminal ends (19).
The cc-chain,which is the most variable of the three chains on
a molecular weight basis(20).,appears to have been less restricted in its evolution at the carboxy-terminus.
d_CHdIN
x-01.12.So.h
1143
OF Hl_?l;tS FIBRISOGES
Phe-Asp-Thr-Ala Pro-
Fig. 6.
.
.
.
Possible internal homologies in the carboxy-terminal portion of the u-chain of human fibrinogen. The data suggest that a series of contiguous duplications 50-60 residues long may have led to elongation of the a-chain cat-boxy-terminal region. Numbering based on Fig. 5.
Plasmin Digestion Points These studies have led
to the identification of two plasmin attack
points near the carboxy-terminal of the a-chains. ages occurs at theLys-Metbond
The first of these cleav-
which corresponds approximately to residues
600-601 in the a-chain (115-116 in Fig. 5).
As we have reported previously
(15),this is apparently the very first plasmin attack point; it also occurs in vivo and leads to separable aichains and aZchains.
A second early plas-
min cleavage occurs at the Lys-Thr bond denoted at residues 48-49 in Fig. 5 (approximately residues 567-568 in the u-chain). Arginyl-Glycine Bonds There are four arginyl-9lycine peptide bonds in this quarter of the a-chain, about ten times more than would be expected on a strictly random basis.
As is well known, thrombin attacks only arginyl-glycine bonds when
releas ing fibrinopeptides, regardless of species. narrow specificity when activating factor XIII
(2!),
It also adheres to this and it will be inter-
esting to find if any of these bonds are cleaved during the thrombincataly zed fibrinolysis which has been reported recently (22).
llttlr
OF HWU?i Of.-CHXI1L‘
Vol.l2,No.6
FIBRIBOGEN
a-Chain Cross-Linking Sites The carboxy-terminal segments of a-chains also contain a portion of the z-chain
multimeric cross-linking system.
We have previously shown that
cyanogen bromide digestion of fully cross-linked fibrin generates a crosslinked polymer comprised of two fragments, one of which (C&I)
stretches
approximately from residues 241-500, and the other of which (CN-IVB)
is
the
penultimate cyanogen bromide fragment in the chain and corresponds approximately to residues 558-607 (14). and only one glutamine.
The latter fragment contains four lysines
Studies are under way to find which of these are
involved in the cross-linking. Status of the Remainder of the a-Chain Sequence At this point approximately two-thirds of the a-chain amino acid sequence has been reported, including the first 272 residues from the aminoterminus (23) and the 142 residues at the carboxy-terminus.
The 210 :!lO
residues which comprise the intervening region all occur in a single cyanogen bromide fragment (CN-I);
the region is distinctive in that it contains
a very large number of serine and glycine residues.
So far we have isolated
and characterized 22 tryptic peptides from this region, and an effort to obtain suitable overlaps by alternative enzymatic digestion is currently under way in order to complete the sequence.
ACKNOWLEDGEMENTS We would like to thank Marcia Riley for preparing fibrinogen and achains,and Dennis Trovato for the operation of amino acid analyzers.
This
study has been supported by USPHS Grants do. HL-18,576 and GM-17,702. REFERENCES 1.
ItiAiiAGA, S., WALLEN, P., GRG:iDAHL, 1'4. J., HENSCHEN, A. AND BLOMBACK, B. On the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digest. Eur.J. Biochem. 8, 189, 169.
2.
TAKAGI, T. and DOOLITTLE, R. F. The amino acid sequences of those portions of human fibrinogen fragment E which are not included inthe anincterminal disulfide knot. Thromb. Res. 7, 813, 1975.
3.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence studies on the achain of human fibrinogen. Location of four plasmin attack points and a covalent cross-linking site. Biochemistry. 14, 5149, 1975.
1145
4.
HARFEttIST, E. J. and CANFIELD, R. E. Degradat ion of fibrinogen by plasi!lirl. IS0lation of an early cleavage product. Biochemistrv. 14, 4110, 1975.
5.
DOOLITTLE, R. F., CASSMAN, K. G., COTTRELL, B. A ., FRIEZNER, 5. TAKAGI, T. Amino acid sequence studies on the a-chain of human gen. Covalent structure of the 1-chain portion of fragment D. 16, 1710, 1977. chemistry.
6.
GARDLUND, B. Human fibrinogen--amino acid sequence of fragment E and of adjacent structure in the Aa- and Es-chains. Thromb. Res. 10, 689, 1977.
7.
DOOLITTLE, R. F., CASSMAN, K. G., COTTRELL, B. A., FRIEZNER, S. J - , HUCKO, J. T. and TAKAGI, T. Amino acid sequence studies on the c(chain of human fibrinogen, Characterization of 11 cyanogen bromide fragments. Biochemistry. 16, 1703, 1977.
J. and fibrinoBio-
MROSS, G. A. and DOOLITTLE, R. F. Step-wise degradation of peptides attached to solid supports. Mol. Biol., Biochem. & Biophys. 25, 1, 1977. 9.
DOOLITTLE, L. R., MROSS, G. A., FOTHERGILL, L. A. and DOOLITTLE, R. F. A simple solid-phase amino acid sequencer employing a thioacetylation stepwise degradation procedure. Anal. Biochem. 78, 491, 1977.
10.
GRAY, W. Sequence analysis with dansyl chloride. 333, 1972.
11.
OFFERD, R. E. Electrophoretic mobilities of peptides on paper and their use in the determination of amide groups. Nature (London). 211, 591, 1966.
12.
HOUMARD, J. and DRAPEAU, G. R. Staphylococcal protease: a proteolytic enzyme specific for glutamyl bonds. Proc. Natl. Acad. Sci., U. S. 69, 3506, 1972
13.
HORN, M. J. and LAURSE:i, R. .A. Solid phase Edman degradation: attachment of carboxyl-terminal homoserine peptides to an insoluble resin. FEBS Letters. 36, 285, 1973.
14.
DOOLITTLE, R. F. , CASSMAN, K. G., COTTRELL, B. A. AND FRIEZNER, S. J, Amino acid sequence studies on the a-chain of human fibrinogen. Isolation and characterization of two linked a-chain cyanogen bromide fragments from fully cross-linked fibrin. Biochemistry. 16, 1715, 1977.
15.
COTTRELL, B. A. and DOOLITTLE, R. F. The amino acid sequence of a 27-residue peptide released from the a-chain carboxy-terminus during the plasmic digestion of human fibrinogen. Biochem. Biophys. Res. Comm. 71, 754, 1977.
16.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence of the carboxyterminal cyanogen bromide peptide of the human fibrinogen s-chain: Homology with the corresponding y-chain peptide and presence in fragment D. Biochim. Biophys. Acta. 386 , 617, 1975.
17.
HENSCHEN, A. and LOTTSPEICH, R. Sequence homology between a-chain and s-chain in human fibrin. Thromb. Res. 1 , 869, 1977.
18.
WATT, K. W. K , TAKAGI, T. and DOOLITTLE, R. F. The amino acid sequence of the a-chain of human fibrinogen: homo ogy with the y-chain. Proc. iIat1.Acad. Sci., U.S. In Press.
Meth. Enzymol. 25,
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19.
DOOLITTLE, R. F. The evolution of vertebrate fibrinogen. Proceedings. 35, 2145, 1976.
Federation
20.
DOOLITTLE, R. F. Structural aspects of the fibrinogen to fibrin conversion. Adv. Prot. Chem. 27, 1, 1973.
21.
TAKAGI, T. and DOOLITTLE, R. F. Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. Biochemistry. 13, 750, 1974.
22.
KANG, E. P. and TRIANTAPHYLLOPOULAS, D. C. Fibrin digestion with thrombin. Comparison with plasmin-digested fibrinogen. Biochim. Biophys. Acta. 490, 430, 1977.
23.
DOOLITTLE, R. F., WATT, K. W. K., COTTRELL, B. A. and TAKAGI, T. Fibrino. gen: A highly evolved regulator agent for maintaining the integrity of the vertebrate circulatory system. Intern. Symp. Proteins, Taipei, 1973. Academic Press. In Press.
