Purification of an almond emulsin fucosidase on Cibacron blue-sepharose and demonstration of its activity toward fucose-containing glycoproteins.

The almond emulsin fucosidase that specifically hydrolyzes fucose in alpha (1-3) linkage to N-acetylglucosamine has been purified 1250-fold. The purification procedure includes ion exchange chromatography on sulfopropyl-Sephadex C-25, gel filtration on Sephacryl S-200, and affinity chromatography on Cibacron blue-Sepharose 4B-CL. The molecular weight of the fucosidase was estimated by gel filtration as approximately 73,000. Enzyme activity was maximal at pH 5.3 in acetate buffer and was dependent on ionic strength; at least 0.1 M NaCl was necessary for optimal activity. The purified enzyme was free of beta-galactosidase activity toward the glycoprotein substrate [3H]galactosyl-asialotransferrin and did not release fucose from substrates containing fucose in alpha (1-6) linkage, (bovine IgG glycopeptides) or in alpha (1-2) linkage, (2'-fucosyllactose). The fucosidase displayed activity toward two glycoprotein substrates known to contain fucose in alpha (1-3) linkage. Extensive incubations resulted in the release of 83% and 43% of the total fucose of asialoorosomucoid and lactoferrin, respectively. The fucosidase did not release fucose from either the "slow" or the "fast" form of alpha 2-macroglobulin, suggesting the absence of fucosyl alpha (1-3) linkages on that glycoprotein.

Interaction of Mycoplasma gallisepticum with sialyl glycoproteins.

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I, a glycoprotein present in articular lubricating fraction of bovine synovial fluid.

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly alpha-(2 leads to 3) to D-galactose residues. The resistance of 2-acetamido-2-deoxy-D-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-1 with alkaline sulfite, approximately 80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal leads to GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-alpha-D-galactosidase (which suggests an alpha-D-GalNAc leads to L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan-Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae beta-D-galactosidase, which hydrolyzes only galactosides linked beta-D-(1 leads to 4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes beta-D-galactosidase released greater than 90% of D-galactose. These results provide evidence for beta-D-Galp-(1 leads to 3)-alpha-D-GalNAcp-(1 leads to 3) alpha-D-GalNAcp-(1 leads to 3)-L-Ser or -L-Thr and alpha-NeuAc-(2 leads to 3)-beta-D-Galp-(1 leads to 3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.

Hepatic receptor that specifically binds oligosaccharides containing fucosyl alpha1 leads to 3 N-acetylglucosamine linkages.

Evidence is presented suggesting that hepatocytes contain a receptor that binds glycoproteins specifically through fucose in alpha1-->3 linkage to N-acetylglucosamine. Human lactoferrin, which contains this type of linkage, is rapidly cleared from the circulation of mice after intravenous injection, and greater than 90% of the injected material is found in hepatocytes. Binding of lactoferrin is mediated through its carbohydrate groups, since its clearance is prolonged after periodate oxidation or after its oligosaccharide groups are extensively degraded with glycosidases. In addition, glycopeptides from lactoferrin inhibit lactoferrin clearance. That lactoferrin clearance is mediated through binding to its fucosyl groups is suggested for several reasons. First, transferrin and asialotransferrin, whose oligosaccharide groups are essentially structurally identical to those of lactoferrin but devoid of fucose, are not cleared on intravenous injection. Second, when fucose is incorporated into asialotransferrin by alpha1-->3 N-acetylglucosamine fucosyl transferase, the resulting fucosylated derivative is cleared rapidly. Neither mannan nor derivatives of orosomucoid that are cleared by binding to receptors for galactose, N-acetylglucosamine, or mannose, inhibit clearance of lactoferrin although clearance is inhibited by fucoidin. Finally, glycoproteins containing fucose in alpha1 --> 2 linkage to galactose or alpha1 --> 6 linkage to N-acetylglucosamine do not inhibit lactoferrin clerance by the liver. Since clearance of other glycoproteins, such as human lactoperoxidase, also appears to be mediated through binding to the same hepatocyte receptor as lactoferrin, it is concluded that the fucose-specific receptor studied here may fulfill other functions than binding lactoferrin. Preliminary studies with liver homogenates and detergent extracts of liver show binding in vitro.