The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E

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Biochimica et Biophysica Aeta, 577 (1979) 110--116 © Elsevier/North-Holland Biomedical Press

BBA 38122

THE CARBOHYDRATE PROSTHETIC GROUPS OF RAT FIBRINOGEN PLASMIC FRAGMENT E G.G. LINDSEY a, G. BROWN a, E.H. M E R R I F I E L D b and L.R. PURVES a,*

a Department of Chemical Pathology, University of Cape Town Medical School, Observatory, Cape Town, 7925, and b Department of Chemistry, University of Cape Town Private Bag, Rondebosch, Cape Town, 7700 (Republic of South Africa) ( R e c e i v e d August 1st, 1978)

Key words: Carbohydrate prosthetic group; Fragment E; Fibrinogen; Fibrin monomers; Plasmin; (Affinity chromatography)

Summary Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per 7-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid -* galactose mannose ~, mannose ~ glucosamine -* glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non
Introduction

Plasmin digestion of fibrinogen yields a 2 : 1 ratio of the two major terminal digestion products, fragments D and E, of molecular weight 80 000 and 50 000, respectively [1]. Most purification methods (summarised in Ref. 1) yield incomplete separation unless carried out under denaturing conditions since fragments D and E are cleaved from fibrinogen partly as a discrete D-E complex held together by hydrophobic interactions [2]. Fragment D contains at least one calcium binding site that protects the C terminus of the ~/-chain against extended plasmin cleavage thus limiting the number of plasmic cleavage products [3]. Fragment E contains the N-terminal regions of all 6 polypeptide chains and is closely related in structure to the cyanogen bromide N-terminal cleavage product called the N-terminal disulphide knot that in addition con* To w h o m reprint requests should be addressed. Abbreviation: SDS, sodium dodecyl sulfate.

111 tains the A and B fibrinopeptides [4]. Both fragments contain carbohydrate prosthetic groups [5]. In fragment E this represents the total amount present on the ~,-chains of fibrinogen [6] and consists of a single oligosaccharide chain attached to each Asp-52 [4]. Differing data have been reported on the carbohydrate content of this oligosaccharide. Mester et al. [7] isolated glycopeptides from a pronase digest of bovine fibrinogen and found that the average fibrinogen glycopeptide contained 11 residues, 4 each of glucosamine and mannose and 3 of galactose. One glycopeptide was found to have originated from the 7-chain. This contained 1 sialic acid and had the galactose residues linked to N-acetylglucosamine; 2 of the mannose residues were 1--6 linked; the other 2 were 1--3 linked. Linkage of the oligosaccharide to the peptide chain was through a glucosamine-asparagine-glycosylamine linkage [7]. These results correlated with those of Bray and Laki [8] who had isolated a glycopeptide from pronase-digested bovine fibrinogen with a sequence, from the nonreducing end, of sialic acid-galactose-N-acetyl glucosamine. For human fragment E, Pepper et al. [5] found the oligosaccharide contained 2 sialic acid, 11 neutral sugar and 4 hexamine residues. Mills and Triantaphyllopoulos [9], however, reported values of 5 neutral sugars and 2.5 hexosamine residues per 7-chain and Blomb~ick et al. [4] reported the presence of 2 glucosamine residues per 7-chain. In this report we describe a nondenaturing method for the purification of fragment E from plasmin digested rat fibrinogen. The carbohydrate content has been investigated and a possible sequence is suggested. Materials and Methods Fibrinogen was purified [3] from the plasma of Long-Evans rats that had been injected subcutaneously with turpentine (4 ml/kg) 48 h prior to exsanguination. Heparin (500 U/l) was used as an anticoagulant. Plasmin digestion of fibrinogen was carried out in the presence of 5 mM Ca2÷ as described previously [10]. The criterion for fragment E purity was a single band after polyacrylamide gel electrophoresis in the presence of SDS. Flat-bed gradient gels (20 X 14 X 0.2 cm) were used throughout. Purification by affinity chromatography was carried out as follows: 2 ml of fibrinogen digest were loaded onto a column (30 X 1.5 cm) of human fibrin-monomer coupled with Sepharose 4B prepared by the method of Kudryk et al. [11]. Elution was with the plasmin digestion buffer (50 mM Tris, 150 mM NaC1, 5 mM CaC12). Fractions (1 ml) were collected and analysed for fragments D and E by polyacrylamide gel electrophoresis in the presence of SD8. Fragment E was finally separated from fibrinopeptide contaminants by Sephadex G-200 gel filtration. The peak containing fragment E was pooled, dialysed against 0.6 M NH4HCO3 and lyophilised. The molecular weight of fragment E was found to be 45 000 as determined by polyacrylamide gel electrophoresis in the presence of SDS with standards of known molecular size. The N-terminal disulphide knot fragment was isolated by the method of B15mback [12]. Thrombin digestion was carried out with bovine thrombin (Sigma) in plasmin digestion buffer. Graded acid hydrolysis, pronase digestion and high voltage paper electrophoresis were carried out by the methods of Spiro [13].

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Sugar analyses on peptides were carried o u t by the anthrone, Warren and Elson-Morgan reactions as described by Spiro [14]. Gas chromatography of the trimethylsilyl ether derivatives of sugars was carried out essentially as described by Sweeley et al. [15] using 3% SE-52 silicone gum adsorbed on Chromosorb W (80--100 mesh) with the oven temperature held isothermally at 150°C. Preparation of peptide samples for GLC sugar analysis was as previously described [14,15]. This consisted essentially of acid hydrolysis under nitrogen followed by coupled chromatography on Dowex 50 H ÷ and Dowex 1 formate. Neutral sugars did n o t bind to either column; hexosamines were eluted from the Dowex 50 H ÷ column with HCI. The eluates were lyophilised prior to derivatisation. Protein concentrations were determined by the m e t h o d of Lowry et al. [16]. Neuraminidase and ~-galactosidase (Sigma) digestion of peptides was carried o u t as described by Spiro [13] and a-mannosidase (Sigma) digestion using the conditions of Levvy and Conchie [17]. Survival times of native and desialated fragment E in the circulation of the rat were determined by the m e t h o d of Morell et al. [18] using material labelled with ~2sI by the m e t h o d of Greenwood et al. [19]. Radioactive fragment E was injected into the tail vein of 250-g rats. The animals were bled from the tail vein at regular intervals and the plasma radioactivty was determined. Results

Chromatography of a fibrinogen digest on columns of fibrin monomerSepharose 4B resulted in the appearance of fragment E in the eluate before fragment D (Fig. 1). Similar results were obtained with a plasmin digest of human fibrinogen thus showing that the reaction was n o t entirely species-specific. Fragment D interacted with the fibrin m o n o m e r and was thus retarded. This step was only efficient if the D molecule had been stabilised with calcium ions during plasmin digestion in order to avoid the production of smaller D species that have no affinity [10]. Fragment E was then purified to homogeneity by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of SDS of ~-mercaptoethanol-treated E showed that only the peptide chain of intermediate molecular weight contained carbohydrate by staining with the periodic acid-Schiff reagent. Human fragment E has chains of moleculax size a > ~, > ~ [20]. Indirect confirmation of the attachment of the carbohydrate moiety to the 7-chain was achieved by isolation of the N-terminal disulphide knot and subsequent thrombin treatment. Thrombin cleaves the A and B fibrinopeptides from the a- and H-chains of the N-terminal disulphide knot respectively. Only the periodic acid-Schiff-positive chain showed no change in molecular weight after thrombin treatment and it is therefore the 7-chain. Gel filtration on Biogel P2 of fragment E that had been exhaustively digested with pronase resulted in only 1 peak of carbohydrate-positive material (Fig. 2) suggesting therefore that only a single type of carbohydrate moiety was present [13]. This peak was positive with the anthrone, Warren and Elson-Morgan reactions, demonstrating therefore the presence of neutral sugars, sialic acid and hexosamines. High voltage paper electrophoresis carried out at 400 V in pyridine/acetic acid buffer (pH 6.4) on Whatman 3MM paper confirmed the pres-

113

E

Fractk~n number

Fig. 1. Fibrin m onomer°Sephar°se 4B chromatography of fibzinogen digested by plasmln in the presence of calcium ions. (A) A280 profile of eluate. (B) polyacrylamide slab gel of samples from the eluate: lane I (left), starting material; lane 2, fraction 19; lane 3, fraction 21; lane 4, fraction 23; lane 5, fraction 27; lane 6, fraction 29; and lane 7 fraction 31. Fractions 18--21 were pooled, concentzated by dialysis against solid polyethyleneglycol and subjected to Sephadex G-200 gel filtration to separate fragraent E from fihrinopeptide contamination seen on the b o t t o m of the gel.

ence of only I glycopeptide. All subsequent work was therefore carried out on whole fragment E in order to minimise losses and to ensure a known molecular weight for the carbohydrate containing peptide. Using a molecular weight for E of 45 000 the chromogenic reactions on whole fragment E revealed (as probable integral amounts} 2 tool sialic acid, 6--8 mol neutral sugars and 4 tool amino sugars per mol fragment E (Table I).

54.9

A 620 O 0.5

02 a 20

5O

80

FRACTION NO Fig, 2. Gel filtration on Biogel P2 of pronase digested fragment E. ~ ; A280" Colorimetric carbohydrate analyses were performed on all fractions bY the anthrone (v- . . . . . v) A260), Wat~en (~- :v) A549 ) and Elson-Morgan reactions (results n o t shown). Positive reactiOnS by all three methods were only found on one peak of material eluting between fractions 26 and 32.

114 TABLE I CARBOHYDRATES Assay

DETECTED

ON FRAGMENT

E

Chromogenic reactions

GLC Sugar

mol/mol fragment E

Probable integral No.

6--8

galactose mannose

2.2 3.6

2 4

4

glucosamine

4.2

4

Detects

mol/mol fragment E

Probable integral No.

Warren

sialic a c i d

1.7

2

Anthrone

neutral sugars

7.3

Elson-Morgan

hexosamine

3.7

Thin-layer chromatography of an acid hydrolysate showed the presence of galactose, mannose and glucosamine. Graded acid hydrolysis showed that the sugars were released in the order galactose, mannose, glucosamine. Treatment of fragment E with 0.5 M NaOH for 24 h at 4°C did n o t release the carbohydrate moiety from the protein as determined by periodic acid-Schiff staining after polyacrylamide gel electrophoresis in the presence of SDS. This suggested a glycosylamine linkage rather than an O-glycosidic or glycosidic ester linkage [13]. GLC of the trimethylsilyl ether derivatives of the individual sugars released b y acid hydrolysis of fragment E confirmed the presence of galactose, mannose and glucosamine. Quantitation by planimetry was in close agreement with the chromogenic methods (Table I). Since fragment E consists of two ~,-chains, each carbohydrate prosthetic group probably contained 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues if exact symmetry between 7-chains was assumed. Incubation of fragment E with neuraminidase resulted in the release of 97% of the b o u n d sialic acid. /~-Galactosidase treatment of neuraminidase-treated material resulted in removal of the galactose moiety as determined by GLC of the trimethylsilyl ether derivatives of the residues. Subsequent ~-mannosidase treatment resulted in the complete removal of neutral sugars. Since these enzymes are exoglycosidases [21] the possible sequence of sugars in the oligosaccharide prosthetic group of fragment E is: sialic acid-~ galactose ~, mannose ~- mannose ~, glucosamine -~ glucosamine -* asparaglne. Neuraminidase treatment of fragment E had no effect on its rate of clearance from the circulation of the rat relative to native fragment E. The half life of native fragment E was found to be 46 min compared with 52 min for desialated fragment E. Discussion We have described a non-denaturing method for the isolation of fragment E from fibrinogen digested by plasmin. Our m e t h o d used the affinity of fragment D for fibrin m o n o m e r as a means of dissociation of the D-E complex. Sufficient calcium ions were present during fibrinogen digestion to prevent smaller noninteracting D molecules being formed that would otherwise have contaminated the fragment E preparation [22].

115 Previous methods used to isolate fragment E free of fragment D have used denaturing conditions viz. gel filtration in the presence of 1 M KI or 10% acetic acid and heat precipitation. Nondenaturing methods, e.g. DEAE-ceUulose ionexchange chromatography yield incomplete separation of these two fragments due to their release from fibrinogen as a D-E complex [2]. Pevikon block electrophoresis has been used for the separation of fragments D and E [23]. However, this m e t h o d has the disadvantages of long running times and a failure to separate fragment D from any undigested fibrinogen. With the fibrin monomerSepharose 4B method, any undigested material and D-dimer from fibrin is bound to the column. Rat fragment E, like h u m a n and bovine, has carbohydrate linked only to the ~/-chain through a glycosylamine linkage. Our data suggests that the oligosaccharide chain is composed of 6 residues, 1 sialic acid, 3 neutral sugars (1 galactose, 2 mannose) and 2 glucosamine. For our calculations we used a molecular weight of 45 000 for fragment E. Fragment E is initially formed as a larger protein and is gradually trimmed by plasmin to its final molecular weight of 45 000 [10,25]. Any difference in the molecular weight will only affect the agreement between carbohydrate content per mol and the probable integral number. Better accuracy is obtained by using a larger protein since incorrect determination of the molecular weight of a glycopeptide will have a larger effect on the carbohydrate molar ratios. The literature generally agrees on the identity of the sugar moieties comprising the oligosaccharide chain on fragment E. Most workers report the presence of sialic acid, with galactose and mannose as neutral sugars and N-acetylglucosamine as the only hexosamine present. The quantity of the individual sugars reported is more varied; values range from 3 neutral and 2 amino sugars per v-chain (this work) to 5--6 neutral sugars and 4 amino sugars per ~,-chain [ 5 ]. These differences may reflect the following factors: (a) Species differences since data has been reported for human, bovine and rat fragment E. (b) Variation in the m e t h o d of sugar estimation as protein interference with chromogenic reactions is well d o c u m e n t e d [14]. It is interesting to note the variation in neutral sugars measured by a chromogenic m e t h o d compared with GLC (Table I); (c) Microheterogeneity in the carbohydrates attached to identical peptide chains. Although this is probably due to postsynthetic but presecretion, nonhomogeneous carbohydrate addition, it could also be caused by subsequent modification [24]. Our sequence data agree with those of Mester et al. [7] and Bray and Laki [8] except that we found galactose bound to mannose rather than to glucosamine. This m a y either reflect a species difference between rat and bovine fibrinogen or glucosaminidase contamination of ~-mannosidase. The latter possibility is unlikely since results obtained by sequential enzymic cleavage and graded acid hydrolysis were in agreement and TLC. of the sugars released by graded acid hydrolysis showed that no free glucosaminine was present until mannose had been released. The possible sequence is sialic acid-~ galactose mannose ~, mannose ~, glucosamine -~ glucosamine -* (Asn}. Removal of the terminal sialic acid had no effect on the clearance rate of

116 fragment E from the circulation of the rat so that no role is played by the carbohydrate prosthetic group in the clearance of fibrinogen degradation products. The clearance and turnover of fibrinogen and fibrin in any event probably mainly takes place outside the vascular compartment by direct cellular pinocytosis. Acknowledgements The authors wish to acknowledge financial support from South African Medical Research Council, the Atomic Energy Board, the Harry Crossley Foundation and the Nellie Atkinson Trust. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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