Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

Abstract

The cell surface glycoprotein ␥-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

“…The only evidence that hGGT2 transcripts are actively translated in vivo is a report in which a single tryptic peptide unique to the amino-acid sequence of hGGT2 was identified during a high-throughput mass spectrometric characterization of human liver glycopeptides (7). However, we did not detect hGGT2-specific peptides within the pool of tryptic fragments from protein immunopurified from normal human kidney and liver with our GGT129 antibody (51,52). This antibody was developed against a peptide that is identical in hGGT1 and all three variants of hGGT2 [ Fig.…”

Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

“…GGTP is found in organisms from bacteria to mammals and plants. Literature data concerning GGTP biochemical properties showed evidence of high thermostability, independence of pH variations, and stability in high-salt conditions (16)(17)(18)(19)(20)(21). AMPs comprise a large family of biologically important zinc enzymes playing different roles in the regulation of blood pressure, antigen presentation, memory, cell cycle, pregnancy, and angiogenesis.…”

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

“…3, E and F). N-glycosylation at Asn-297 occurred in hGGT1 isolated from human tissues but was absent in the crystal structure of the yeast-expressed hGGT1 (35).…”

“…Previous studies on the N-glycosylation pattern of hGGT1 isolated from human tissues identified seven N-glycosylation sites (35,36). Because we trimmed the glycans to the proximal glycan with Endo H f before crystallization, the N-glycosylation sites were marked by one GlcNAc residue.…”

“…Finally, hGGT1 has different requirements for autocleavage compared with bacterial GGTs. hGGT1 has seven N-glycosylation sites, and proper folding and autocleavage of the propeptide requires co-translational N-glycosylation (35,36). The bacterial GGTs with crystal structures lack N-glycosylation, yet their propeptides are properly folded and undergo autocleavage (27)(28)(29)37).…”