Glycoprotein biosynthesis: The basic protein encephalitogen from bovine spinal cord, a receptor for the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands

Abstract

A basic protein from bovine myelin, which contains no carbohydrate, was found to serve directly as a natural receptor for the N-acetylgalactosamine transferred from UDP- N-acetylgalactosamine (UDP-galNAc) by the polypeptidyl: N-acetylgalactosamine transferase of bovine submaxillary glands. This receptor, which mimicked in all respects the “artificial” receptor prepared from the bovine submaxillary glycoprotein by removal of the N-acetylhexosamine with hexosaminidase, possessed approximately 25% of the receptor activity (cpm galNAc- 14C incorporated per mg protein) when compared to the latter receptor. Using either receptor, the transfer reaction requires Mn 2+ ion. The product formed in both cases involves a glycosidic linkage between N-acetylgalactosamine- 14C and hydroxyamino acids; the galNAc- 14C was released from the basic protein- 14C product by hexosaminidase and N-acetylgalactos-aminitol- 14C was isolated after alkaline borohydride treatment. The importance of receptor size as a requirement for receptor function was revealed by the loss of activity following Pronase digestion of the A1 protein. The relative stability of both receptors to heating at 100 °, or 8 m urea treatment, suggested that the reactive site of the receptors is primarily dependent on the requirements of a polypeptide sequence rather than tertiary structure. The results of this work support and extend the conclusions derived from study of the specificity of the receptor requirements using the submaxillary protein as a receptor for the submaxillary gland transferase. The basic protein- 14C material was degraded with proteolytic enzymes; two of the radioactive peptides obtained following purification on Dowex 50 resin and high-voltage paper electrophoresis revealed that a threonine was joined in these peptides in an O-glycosidic linkage to galNAc- 14C. It was concluded that a polypeptide sequence exists in the A1 protein, containing a functional threonine residue, that meets the receptor requirements of the polypeptidyl: galNAc transferase as found in the submaxillary glycoprotein.