Glycoprotein biosynthesis: Studies on the receptor specificity of the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands

Abstract

The receptor requirements of the polypeptidyl: N-acetylgalactosaminyl (galNAc) transferase, purified from bovine submaxillary glands, was investigated. The transfer of galNAc- 14C to the hydroxyamino acid residues of the polypeptide chain of submaxillary glycoprotein denuded of carbohydrate is a highly specific process. All other proteins and glycoproteins studied, intact and partially degraded, were nonfunctional in this reaction except for the submaxillary polypeptide. Receptors were obtained from desialized bovine submaxillary glycoprotein (BSM) by three procedures: (1) removal of the N-acetylhexosamine residues by epididymal hexosaminidase; (2) periodate treatment followed by reduction with NaBH 4 and mild acid hydrolysis; and (3) directly by acid hydrolysis (0.2 n H 2SO 4, 2 hr 100 °). Up to 17–22% of the N-acetylhexosamine previously removed could be replaced by the galNAc- 14C using the purified transferase. The best receptor material was obtained by removal of 50–70% of the N-acetylhexosamine by hexosaminidase or the periodate procedure. A linear relationship was found between the percentage of N-acetylhexosamine removed by hexosaminidase, up to approximately 50%, and the incorporation of galNAc- 14C. When more than 70–75% of the N-acetylhexosamine had been removed, however, no further increase in receptor activity was found; upon removal of 75% or more of the N-acetylhexosamine, the receptor activity was significantly decreased. Materials such as intact BSM or desialized BSM, from which the N-acetylhexosamine had not been removed, were nonfunctional as receptors. The hydroxyl groups of sugar residues would not substitute for those of specific hydroxyamino acids of the submaxillary polypeptide chain. In this respect, N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid were all nonfunctional as receptor sites as demonstrated by the inactivity of bovine and porcine submaxillary glycoproteins, the α 1-glycoprotein, and fetuin, each of which had been partially degraded with specific glycosidic enzymes. The specificity of the reaction for particular hydroxyamino acids of the BSM polypeptide receptor was inferred by the inactivity of the submaxillary glycoprotein after alkali-borohydride treatment. Twenty-five proteins, native and denatured, were tested for receptor activity with negative results. The only material 2 2 After this study had been completed, it was found that the basic protein encephalitogen, a homogeneous protein isolated from bovine spinal cord, had approximately 25% of the receptor activity of the bovine submaxillary polypeptide from which 50% of the N-acetylhexosamine had been removed (1). with receptor activity was found in the extract of the submaxillary glands and most likely represents endogenous BSM material at incomplete stages of synthesis. The receptors prepared from BSM exhibited both conformational and size requirements although they were unusually stable to some conditions known to denature proteins. In spite of treatment with 0.2 n H 2SO 4, 90 °, 2 hr, or with 65% ethanol, receptor activity was not affected. After the use of 8 m urea or heating at 100 °, 5 min, approximately 50% of the receptor activity still remained. The size requirements of the hydroxyamino acid-containing polypeptide chain was illustrated by the complete loss of receptor activity after Pronase digestion. After trypsin treatment, which yields larger peptides than Pronase, 35% of the receptor activity still remained. Small molecules, such as serine, threonine, triserine, trithreouine, and a pentapeptide containing both serine and threonine were also inactive. The results from this work emphasize the high specificity required of the gly cosyl transferases in general for synthesis of the specific structures found in the oligosaccharide units of glycoproteins. The existence of the polypeptidyl: galNAc transferase is consistent with a proposed “one enzyme-one linkage” concept of glycoprotein biosynthesis.