Analysis of site-specific glycosylation of renal and hepatic γ-glutamyl transpeptidase from normal human tissue

Abstract

The cell surface glycoprotein γ-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.

FIGURE 1.

Electrophoresis of GGT from human kidney and liver microsomes. A, native gel analysis of GGT. Triton X-100-solubilized microsomes from normal human kidney (lanes 1–3 and 7–9) or liver (lanes 5 and 6 and 11 and 12) tissue were incubated at 37 °C with papain alone (lanes 1–6) or with papain and clostridial neuraminidase (lanes 7–12) and resolved in parallel on 10% nondenaturing polyacrylamide gels. GGT was localized in situ with the histochemical stain for GGT activity (red precipitate). B, SDS-PAGE analysis of intact and deglycosylated GGT. Non-papain-treated samples from human kidney (lanes 1 and 2 and 6 and 7) and liver (lanes 3 and 4 and 8 and 9) were denatured and incubated in the absence (lanes 1–4) or presence (lanes 6–9) of PNGase F and resolved on an 8% (upper panel) or 10% (lower panel) SDS-polyacrylamide gel. Resolved proteins were electroblotted onto nitrocellulose and Western blotted using antibodies against the large and small subunits of GGT. Positions of molecular mass (M) markers are indicated.

FIGURE 2.

MALDI-TOF-MS glycomic profile of permethylated N-glycans derived by PNGase F digestion of a soluble detergent extract of membranes from the cortex of normal human kidney 1 (A) and GGT purified from human kidney 1 (B) are shown. B, inset, the immunopurified large subunit of kidney GGT used for glycomic analysis was analyzed by SDS-PAGE and stained with Coomassie Blue (E, sample; M, molecular mass markers). Symbols used are as follows: blue box, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; half-green and half-yellow circle, hexose (galactose or mannose); red triangle, fucose; purple diamond, N-acetylneuraminic acid.

FIGURE 3.

Tryptic glycopeptide sequence map for the large subunit of human GGT. Predicted tryptic glycopeptides are underlined. Arrowheads denote the positions of putative N-glycosylation sites.

FIGURE 4.

Glycopeptide analysis of GGT isolated from normal human kidney cortex. A, base peak intensity chromatogram of the tryptic digest of GGT immunopurified from normal human kidney. The shaded regions depict the retention times of the different glycopeptides associated with this glycoprotein, and Table 1 summarizes the microheterogeneity at each site. B, averaged LC/Orbitrap-FT MS spectrum for the GGT Asn-297 (GYNFSR) family of glycopeptides from kidney tissue, showing the identified glycoconjugates within the elution interval. Asterisks denote glycoconjugates that were confirmed by tandem MS analysis. P represents the GYNFSR tryptic peptide to which the N-glycans are attached. Unannotated peaks represent nonglycosylated peptides that co-eluted with this glycopeptide family. C, MS/MS spectrum showing the fragmentation pattern of the monosodiated glycopeptide at m/z = 1131.7 identified on the Asn-297 glycosylation site. For symbol definitions, see Fig. 2.

FIGURE 5.

MALDI-TOF-MS glycomic profile of permethylated N-glycans derived by PNGase F digestion of a soluble detergent extract of membranes from normal human liver 1 (A) and GGT isolated from normal human liver 1 (B). For symbol definitions, see Fig. 2.

FIGURE 6.

Glycopeptide analysis of GGT isolated from normal human liver tissue. A, base peak intensity chromatogram of GGT tryptic digests immunopurified from liver tissue. The shaded regions depict the retention times of the different glycopeptides associated with this glycoprotein. Table 2 summarizes the microheterogeneity of each site. B, averaged LC/Orbitrap-FT MS spectrum for the liver GGT Asn-344 (NMTSEFFAAQLR) glycopeptide, showing the associated glycoconjugates within the elution interval. Asterisks denote glycoconjugates that were confirmed by MS/MS. Peaks annotated by Φ represent members of the Asn-120 (N-sequon = NSS) family of glycopeptides that co-eluted over this same interval. P represents the NMTSEFFAAQLR tryptic peptide to which the N-glycans are attached. Unannotated peaks represent unmodified peptides that co-eluted over this interval. C, MS/MS spectrum showing the fragmentation pattern of the Asn-120 (NSS) glycopeptide at m/z = 1347.2. For symbol definitions, see Fig. 2.

FIGURE 7.

Tissue-specific N-glycosylation patterns on the Asn-120 site of GGT. The relative abundances of distinct N-glycan structures on the Asn-120 (NSS) glycosylation site, as inferred from glycopeptide ion intensities, are graphically depicted for GGT immunopurified from either kidney (A) or liver (B) tissue. For symbol definitions, see Fig. 2.

FIGURE 8.

Human GGT homology model. A, ribbon diagram of human GGT (large subunit, blue; small subunit, green). Glycosylated asparagine residues are highlighted with CPK atom coloring, and their positions in the amino acid sequence are indicated with white numbers. The position of the substrate channel and catalytic threonine (T381, red sticks) are shown. The homology model is based on the crystal structure of soluble GGT from E. coli. The first 34 amino acids of human GGT contain the transmembrane domain and do not have homology to E. coli GGT. The position of amino acid 35 (aa35) is shown, which defines the orientation of the enzyme on the cell surface. B, space-filling model of human renal GGT with the most abundant N-glycans identified at each site (Table 1). Rotational view of the renal heterodimeric enzyme, featuring a view from the intracellular perspective (i.e. base of transmembrane stalk). The Asn-511 glycosylation site is located on the distal surface of GGT and is obscured by the N-glycan at position Asn-344.