Membrane bound γ-glutamyltranspeptidase: Its structure, biosynthesis and degradation

Abstract

γ-Glutamyltranspeptidase is a glycoprotein composed of heavy and light subunits and associated with the brush border membrane of the kidney and small intestine. γGTP solubilized with papain is a hydrophilic enzyme which has lost the membrane binding segments but its catalytic activity is not altered, whereas γGTP solubilized with Triton X-100 is a hydrophobic enzyme which contains hydrophobic domain binding to the membrane. Amino acid compositions of these two forms were compared and Triton solubilized enzyme was found to contain 52 amino acid residues more than the papain solubilized form. This difference is due to the heavy subunit not light subunit. Then, end group analysis was carried out and the carboxyl-termini of their light subunits were found to be phenylalanine and those of their heavy subunits were tyrosine, respectively. Although light subunits of two forms contain a common sequence, Thr-Ala (X)-Leu as an amino-terminal portion, that of heavy subunit of Triton X-100 solubilized form contains the sequence Met-Lys-Asn-Arg-Phe-Leu-Val-Leu-Gly-Leu-Val-Ala-Val-Val-Leu-Val-Phe-Val-Ile-Ile-Gly-Leu and the papain-solubilized form contains completely different amino-terminal sequence Gly-Pro-Pro-Leu. It is concluded that an amino-terminal portion of the heavy subunit is a hydrophobic domain consisting of about 20 hydrophobic amino acids and contributes to anchor the enzyme to the membrane. γGTP has been known to show great heterogeneity in charge and multiple forms with different isoelectric points are found to be mainly due to differences of their sugar chains. Then the structures of the oligosaccharides attached to γGTP were determined. They were found to be all asparagine linked and consisted of neutral and acidic oligosaccharides with remarkable heterogeneity. A correlation between the contents of the acidic oligosaccharides and the isoelectric points of multiple forms of γGTP was observed. In addition, multiple forms of γGTP were immunologically identical and their protein structures were identical. Next, the mechanisms of biosynthesis of γGTP were examined and it was found that two subunits of γGTP are synthesized as a precursor protein with a single polypeptide chain of 78,000 daltons. Then processing by limited proteolysis occurs post-translationally, and it is a rather slow process. Since the precursor form is already core glycosylated and fucosylated, proteolytic processing could be carried out after completion of terminal glycosylation at the Golgi membrane or the plasma membrane. Moreover, the turnover rates of the heavy and light subunits were the same with half lives of 4.3 days. Thus, although two subunits of γGTP bear a different function, they are synthesized and degraded together. On the other hand, γGTP and aminopeptidase M are closely associated with the microvillus membrane of kidney and small intestine, whereas the two enzymes turn over independently. It suggests that biosynthesis and the degradation of the microvillus enzymes are not carried out as a membrane unit; each enzyme has its own turnover rates.