Primary structure of two major glycans of bovine fibrinogen

Abstract

After pronase digestion of bovine fibrinogen, the asparagine-linked glycans were released from the resulting glycopeptides by hydrazinolysis, and subsequently re-N-acetylated. Two sialylated glycans wcrc isolated by ionexchange chromatography. Their primary structure has been determined by mcthylation analysis and 360-MHz 'H-NMR spectroscopy. The structures proposed to be present in the native glycoprotein are as follows:Fibrinogen and fibrin from a number of animal species are glycoproteins [I]. The biological role of their glycan moieties is not yet clear despite active investigations in this field. For example, it has been reported that neutral carbohydrates [2] or sialic acid [3] are released during clotting but a number of investigations were unable to confirm these results [4, 51. The decrease in coagulability of fibrinogen after periodate oxidation of glycans also suggested a role of the latter in the clotting process [6]. This finding has since been critized, as the reduction of the clotting time may be due to oxidation of some amino acid residues [7]. Recently, it was demonstrated that the enzymatic removal of glycans does not change the clotting time of fibrinogen [8].Study of pathological cases did not bring more information about the relationship between sugars and clotting. In fact congenitally abnormal fibrinogens [9 -1 I] have in some ciiscs a lower carbohydrate content than normal ones but is seems that the primary cause of these coagulation disturbances is an amino acid substitution. However, sialic-acidfree fibrinogen forms a gel different from the normal protein [I21 and yields urea-soluble fibrin in thc presence of factor XI11 [13]. Thesc findings suggest that the carbohydrate in fibrinogen could play a role in the complex polymerisation process.Attempts to clarify the structure of the glycan of bovine fibrinogen were done earlier by Mester et al. [ 14, IS], but due to the poor performances of the analytical methods used at that time, the authors did not obtain exact determination of the structure. The present paper describes the primary structure of the two major glycans of bovine fibrinogen deter-('orre.s/7:llonc(c./~~f~ to

An improved method for quantitative sugar analysis of glycoproteins

“…Note; The values were based on the results from the hydrolyses with 6 M HCl at 80 o C for 2 h. a Taken from Debeire et al (1985). b Taken from Dorland et al (1981).…”

“…Decomposition of neutral sugars would be reflected sensitively in the molar ratio of neutral sugars to amino sugars when oligosaccharides having more neutral sugars than amino sugars are hydrolyzed. Fibrinogen contains a complex type oligosaccharide, Gal 2 -Man 3 GlcNAc 4 (Debeire et al, 1985) and soybean agglutinin contains a high-mannose type oligosaccharide, Man 9 GlcNAc 2 (Dorland et al, 1981). When the two glycoproteins were hydrolyzed under the new hydrolysis condition, the ratio between the neutral and amino sugars were 1.8 ± 0.2 : 2.5 ± 0.1 : 3.6 ± 0.3 and 0 : 7.6 ± 0.9 : 2.0 ± 0.1 (Gal : Man : GlcN) for fibrinogen and soybean agglutinin, respectively (Table 3), which were similar to the theoretical values.…”

Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F

“…In human fibrinogen the ratio of the disialo glycan relative to the monosialo glycan is approximately 2 : 1 [23], whereas in bovine fibrinogen this ratio appears to be 1 : 1 [24]. In human and bovine fibrinogen the carbohydrate chains occur exclusively on the ,fl and y subunits, each subunit bearing one oligosaccharide chain.…”

Conformational Studies of Glycopeptides by Energy Transfer

“…Preparation of Glycopeptides-For large-scale preparation of biantennary glycopeptides, bovine fibrinogen was the most suitable source, because it contains only biantennary-type oligosaccharides (11). Glycopeptides were generated by exhaustive Pronase digestion of reduced and alkylated bovine fibrinogen.…”

“…In order to extend such studies to biantennary glycopeptide, we have chosen to use the major monosialylated biantennary glycopeptide derived from readily available bovine fibrinogen of the structure shown in Fig. 1 (11).…”