Analysis of the C-terminal structure of urinary Tamm-Horsfall protein reveals that the release of the glycosyl phosphatidylinositol-anchored counterpart from the kidney occurs by phenylalanine-specific proteolysis.
Tamm-Horsfall protein (THP), also known as uromodulin, is a major glycoprotein synthesized in the kidney. THP is expressed on the luminal surface of the membrane with the glycosyl phosphatidylinositol (GPI) anchor and excreted in urine at a rate of 50-100 mg per day. Although THP is the most abundant urinary protein, the function of THP remains unclear. In addition, little is known about the mechanism by which large amounts of THP are actively released into the urinary fluid. In this study, we examined the C-terminal structure of highly purified THP derived from human urine. Carboxypeptidase Y efficiently degraded urinary THP, indicating that the C-terminal structure of the protein contains an amino acid residue with a free carboxyl moiety. These results are consistent with our previous finding that urinary THP does not bind anti-CRD antibody. We obtained peptides from the complete digestion of urinary THP with lysylendopeptidase. We purified the most C-terminal peptide with p-phenylene diisothiocyanate-controlled pore glass (DITC-CPG) beads. N-terminal sequence analysis indicated the peptide begins with Tyr 520 and ends between E539 and E576. Direct C-terminal amino acid sequencing of highly purified urinary THP gave a sequence of -X-(Q)-G-(R)-F, corresponding to amino acids 544-548, -S-Q-G-R-F. We therefore conclude that urinary THP is generated by a proteolytic cleavage between F548 and S549, 66 amino acids upstream of a possible GPI-anchor attachment site. Because the sequence of THP, including the cleavage site, is highly homologous to that of GP2, a GPI-anchored protein within the pancreas, and both THP and GP2 are abundantly found as soluble forms in the excreted fluids, a common mechanism may exist governing the proteolytic release of GPI-anchored membrane proteins.
Fukuoka
S
S
Graduate School of Agriculture, Kyoto University, 611-0011 Uji, Kyoto, Japan. fukuoka@food2.food.kyoto-u.ac.jp
Kobayashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009088:Mucoproteins / Q000737:chemistry* / Q000235:genetics / Q000652:urine*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010649:Phenylalanine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D058949:Uromodulin
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009088:Mucoproteins; D010446:Peptide Fragments; C548612:UMOD protein, human; D058949:Uromodulin; D010649:Phenylalanine; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A