Membrane bound gamma-glutamyltranspeptidase: its structure, biosynthesis and degradation

gamma-Glutamyltranspeptidase is a glycoprotein composed of heavy and light subunits and associated with the brush border membrane of the kidney and small intestine. gamma GTP solubilized with papain is a hydrophilic enzyme which has lost the membrane binding segments but its catalytic activity is not altered, whereas gamma GTP 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. gamma GTP 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 gamma GTP 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 gamma GTP was observed. In addition, multiple forms of gamma GTP were immunologically identical and their protein structures were identical. Next, the mechanisms of biosynthesis of gamma GTP were examined and it was found that two subunits of gamma GTP 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.(ABSTRACT TRUNCATED AT 400 WORDS)