Polypeptide-Polysaccharide Conjugates Produced by Spontaneous Non-Enzymatic Glycation

Abstract

A fundamental dogma has developed over the past 20 years that non-enzymatic glycation involving saccharide chains of greater than 3 to 4 residues is an extremely unlikely reaction. Our investigations using glycosaminoglycans have shown that, given sufficient time, polypeptide-polysaccharide conjugates form via the Schiff base-Amadori rearrangement mechanism. Further, even though these straight chain polysaccharides are relatively charged and sterically hindered, spontaneous glycation can also occur in vivo. A complete reinvestigation of all aldose terminating polysaccharides is required to elucidate this new class of macromolecules, which is likely to contain unusual polypeptide-polysaccharide combinations and functions.

Mechanisms Responsible for Catalysis of the Inhibition of Factor Xa or Thrombin by Antithrombin Using a Covalent Antithrombin-Heparin Complex

“…HЈ chains were dialyzed exhaustively against H 2 O and freeze-dried. Analysis of acid-hydrolyzed HЈ on the Beckman System 6300 High Performance Analyzer showed only a large free glucosamine peak and one unidentified peak (likely the lysyl-uronic acid linkage group reported previously (12)). To quantitate the HЈ amino acid end group, weighed out samples of HЈ (in 2 ml of H 2 O) were incubated with ninhydrin reagent (0.2 ml of a solution prepared from 0.2 g of ninhydrin ϩ 7.5 ml of 2-methoxyethanol ϩ 2.5 ml of 4 M sodium acetate, pH 5.5, ϩ 10 mg of SnCl 2 ⅐2H 2 O with constant stirring and bubbling of a stream of commercial grade N 2 ) at 100°C for 15 min.…”

“…Elution was with 0.15 M NaCl, and 1-ml fractions were collected (void volume ϭ fraction 11). Heparin chains (fractions [12][13][14][15][16][17][18][19][20] were well separated from complex-type Asn-linked glycans (fractions 26 -31) or free amino acids (fractions [35][36][37][38][39][40][41][42][43][44][45], as confirmed by control digests of uncomplexed antithrombin. Eluted material was detected by a refractive index detector and measurements given as refractive index units (RIU) relative to 0.15 M NaCl in the reference cell.…”

“…This finding was surprising given that covalently linked AT and heparin were unable to completely dissociate after formation of inhibitor complexes by direct reaction with factor Xa or thrombin (11,12). One possible mechanism that might explain the catalytic activity of ATH was the presence of a second ATbinding pentasaccharide sequence on the covalently linked heparin chain that was separate from the one that activates the conjugate's own AT moiety.…”

“…Previously, we produced a covalent AT-heparin complex (ATH) to further study the mechanism of enzyme inhibition by H-activated AT (11,12). Surprisingly, although serpin and GAG cannot dissociate, it was observed that ATH could catalyze the inactivation of thrombin (or factor Xa) by added AT (11).…”

“…Although binding to thrombin is through nonselective interaction of negative charges on the GAG with the anion-binding exosite of the enzyme (7, 8), AT binding to H occurs through high affinity to a specific pentasaccharide sequence on the heparin chain (2, 9). Moreover, it has been shown that the rate-determining step for catalysis of thrombin (or factor Xa) inhibition involves the initial binding of AT and H (10).Previously, we produced a covalent AT-heparin complex (ATH) to further study the mechanism of enzyme inhibition by H-activated AT (11,12). Surprisingly, although serpin and GAG cannot dissociate, it was observed that ATH could catalyze the inactivation of thrombin (or factor Xa) by added AT (11).…”