Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H.
We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose beta-(1-4)-N-acetylglucosamine beta-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid alpha-(2-6) terminally linked.
Gambino
R
R
Dipartimento di Medicina Interna, Università di Torino, Italy.
Ruiu
G
G
Pagano
G
G
Cassader
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004076:Digoxigenin; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry*; D053482:beta 2-Glycoprotein I
D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D053482:beta 2-Glycoprotein I; D004076:Digoxigenin