Immunochemical studies of extracellular glycoproteins (X-GPs) of goldfish brain

Abstract

Exoglycoproteins (X-GPs) are a family of soluble glycoproteins which are the most prominent constituent of the extracellular compartment of goldfish brain. On conventional two-dimensional polyacrylamide gels they typically display two primary molecular weight forms, averaging about 33 and 38 kDa, each appearing as a row of five to seven individual spots. When X-GP antibodies were applied by Western blotting, gels of goldfish brain extract prepared without a reducing agent showed, in addition to the primary molecular weight groups, at least one row of spots of slightly lower molecular weight and a major array of spots in the range of 45–60 kDa. The latter presumably represent dimers of the primary X-GP forms since they gave rise to the primary forms upon treatment with a reducing agent. However, on gradient gels prepared without detergents or reducing agents, X-GPs identified by immunostaining appeared only at 200 kDa and above, indicating that these proteins naturally occur in the form of large particles. Deglycosylation of the brain extract by N-glycosidase F reduced the molecular weight of each primary X-GP form by about 5 kDa, but did not abolish the microheterogeneity, which is at least partly due to minor differences in primary structure among the proteins in individual spots. Both rows of spots in the deglycosylated sample showed a coordinated shift toward the basic side of the gel, and a prominent new spot appeared on the basic end of the lower molecular weight group, which probably represents the fully deglycosylated form of the most abundant X-GP isoform. The X-GPs were found to be heparin-binding proteins, which is also characteristic of a number of substrate-binding proteins of great physiological importance, including apolipoproteins. However, there was no immunoreactivity with antibodies to apolipoproteins A-I, D, or E, and conversely, antibodies to X-GPs did not react with apolipoproteins from rat serum or from regenerating rat sciatic nerve. Moreover, the X-GPs were not retained in the floating lipid fraction upon centrifugation in high density medium and did not cofractionate with lipids when they were isolated by gel chromatography. Thus they are unlikely to be lipid-binding proteins.