Two forms of
alpha 1-acid glycoprotein with common immunological determinants and almost identical
amino acid compositions but different amounts of
carbohydrate were isolated from liver
metastases of primary colon, lung, and
breast tumors by extraction with
perchloric acid, gel filtration on
Sepharose CL-6B and
Sephadex G-200, and affinity chromatography on
concanavalin A:
agarose and
Ricinus communis agglutinin l:
agarose. Both forms of the
antigen yielded single bands which stained for
protein and
carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six
oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the
oligosaccharide chains in the two forms of this
glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type
oligosaccharide chains. The diantennary
oligosaccharide chains caused both forms of
alpha 1-acid glycoprotein to be retained by
concanavalin A-
agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis
agglutinin I. Three types of reduced
oligosaccharides were released from the light and heavy forms of
alpha 1-acid glycoprotein by treatment with alkaline
borohydride or by hydrazinolysis. These chains were isolated by chromatography on
concanavalin A:
agarose and Bio-Gel P-6 columns. The arrangement and linkage of
sugars in the purified
oligosaccharides were determined by
periodate oxidation, sequential hydrolysis with
glycosidases, and methylation analysis. The major
oligosaccharide chain, comprising 50 to 55% of the
carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is
N-acetylneuraminic acid, Gal is
galactose, GlcNAc is
N-acetylglucosamine, Man is
mannose, GlcNAcol is
N-acetylglucosaminitol, and Fuc is
fucose. Tetraantennary chains comprised about 25 to 30% of the
carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the
oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from
tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the
glycoprotein isolated from liver
metastases.