Rats treated with daily injection of DL-
isoproterenol for 10 consecutive days (25 mg kg(-1)
body weight) showed marked induction of a
proline-rich
glycoprotein (GPRP) of 220 kDa.
Proteinase K digestion of GPRP produced a homogeneous
glycopeptide with an average chemical composition as follows (residues per mol): Pro4, Glx3, Asx2, Gly1, His1, Thr1, Arg1, GlcNAc5, GalNac1, Man3, Gal2-3, and Fuc1. The structural analysis of the
asparagine-linked
carbohydrate unit was performed by methylation,
periodate oxidation and enzymatic degradation. Methylation studies indicated that the three mannosyl residues were substituted at 1,2-, 1,2,4-, and 1,3,6-positions.
Fucose, N-
acetylgalactosamine, 1.5 residues of
galactose and 0.35 residues of
N-acetylglucosamine were terminally located and one
galactose residue was 1,4-substituted. Approximately four of the 5
N-acetylglucosamine residues were substituted at 1,4-position and approximately 1 residue of
N-acetylglucosamine was substituted at 1,4,6-positions.
Periodate oxidation studies and
exoglycosidase results were consistent with the methylation data. Based on the results of Smith degradation, methylation and sequential
exoglycosidase digestions a triantennary
oligosaccharide structure having terminal N-
acetylgalactosamine in one of the branches is proposed for the major Asn-linked
carbohydrate moiety of GPRP.