Characterization of the carbohydrate structures of apolipoprotein H through concanavalin A affinity chromatography

Abstract Apolipoprotein H, also known as β2-Glycoprotein I, is a single chain highly glycosylated polypeptide of 326 amino acids. The carbohydrate content of apolipoprotein H is approximately 19% of the molecular weight. Some studies have described the main oligosaccharides forming the glycosylated chains but the carbohydrate inner structures of apolipoprotein H has not been investigated yet. This gap should be filled being glycosylation a very important process which is able to regulate the structure and the biological functions of proteins. Lectins are proteins which specifically bind carbohydrate structures. Affinity chromatography of glycoproteins on immobilized lectins, such as Concanavalin A (Con A), has been proved to be a useful method for oligosaccharide fractionation. N-Linked oligosaccharide structures were shown to interact with Con A according to their branching properties. In the present study, we analyzed the patterns of Con A elution of apolipoprotein H isolated from human plasma. Using Con A affinity chromatography we show that apolipoprotein H has a high degree of heterogeneity in its glycosylated structure. It allowed one to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Since Con A affinity chromatography allows fractionation of molecules differing in the extent of carbohydrate branching irrespective of the sialyl residues, we can conclude that mannose residues are masked with other sugars such as galactose-/gb(1–4) N -acetylglucosamine, galactose-β(13) N -acetyl-galactosamine and sialic acid linked α-(2–6) to galactose or to N -acetylgalactosamine, or capped with sulfated residues. Thus, according to our results apolipoprotein H presents truncated hybryd or hybrid-type carbohydrate chains which bear few unmasked mannose residues as terminal sugar. Moreover, isoelectrofocusing of apolipoprotein H forms fractionated on Con A demonstrates that weakly bound material presents a predominanceof more acidic isoforms than that firmly bound to the lectin, indicating that weakly bound fractions contain molecules which are more negatively charged and that Con A is able to separate glycosylated forms which are not discriminated by isoelectrofocusing.