Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Abstract

Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.

The Mushroom Marasmius oreades Lectin Is a Blood Group Type B Agglutinin That Recognizes the Galα1,3Gal and Galα1,3Galβ1,4GlcNAc Porcine Xenotransplantation Epitopes with High Affinity

“…Indeed, as shown in Fig. 5, MOA reacted strongly with laminin, which we have shown contains Gal␣1,3Gal␤1,4GlcNAc end groups (22,23), as well as with bovine thyroglobulin, which has the same determinants (24). Significantly, the lectin did not give a precipitin reaction with pigeon ovalbumin, which contains multiple Gal␣1,4Gal end groups (25), thus demonstrating its specificity for Gal␣1,3Gal groups.…”

“…This highly glycosylated glycoprotein contains the linear type B blood group trisaccharide at many of its chain ends (22,23). It also recognizes and precipitates bovine thyroglobulin, which also contains the linear B trisaccharide (24).…”

Carbohydrate Binding Specificity of the Recombinant Chitin-binding Domain of Human Macrophage Chitinase

“…18) Laminin contains 12-15z carbohydrate and most of the carbohydrate is present in complex-type N-linked oligosaccharides. [19][20][21] However, since the structural studies of extracellular matrix glycoprotein carbohydrates are still in progress and the carbohydrate structure varies with the tissue source of the molecule, it is possible that some extracellular matrix molecules contain hybrid-type chains. It is interesting to note that laminin has poly-N-acetyllactosamines attached to N-linked oligosaccharides.…”

“…It is interesting to note that laminin has poly-N-acetyllactosamines attached to N-linked oligosaccharides. 21) Poly-Nacetyllactosamine is a polysaccharide having Nacetyllactosamine (Galb1-4GlcNAc) repeats and structurally similar to chitin and hyaluronan in regard to the presence of GlcNAc residues. Extracellular matrix glycoproteins having poly-N-acetyllactosamine chains, such as laminin could be a potential ligand for the chitin-binding domain of human macrophage chitinase.…”

Galectins: Structures, Binding Properties and Function in Cell Adhesion

“…N-glycans are among the main binding partners of galectin-1, -3 and -8 (Patnaik et al, 2006) (although galectin-8 also shows high affinity to some glycosphingolipids (Ideo et al, 2003;Yamamoto et al, 2008)). One third of laminin Nglycans is composed of repetitive "N-acetyllactosamine" units (shown for mouse EHSlaminin) which are preferentially recognised by galectin-3 but also by galectin-1 and to less extent galectin-8 (Arumugham et al, 1986;Hirabayashi et al, 2002;Knibbs et al, 1989;Sato & Hughes, 1992;Zhou & Cummings, 1993). The other ECM-glycoproteins carry also Nglycans but are less glycosylated (Bunkenborg et al, 2004;Chen et al, 2009;.…”