Abstract
N-Glycans are modified by various glycosyltransferases in a protein-selective manner and critically control protein functions. Here, we investigated the mechanisms for the protein substrate selectivity of N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) in the mouse kidney. Through lectin-assisted proteomics, two metalloproteases on the apical surface of tubules, alanyl aminopeptidase (ANPEP) and meprin α (MEP1A), were identified as the major GnT-V substrates. We further determined the GnT-V-modified glycosites and revealed that the modified sites are highly accessible and clustered within the C-terminal domains, suggesting some structural requirements for GnT-V action. Single-cell transcriptomics revealed partial overlap of Mgat5 and Anpep or Mep1a, excluding the possibility that Mgat5 is limited in the same cell type as the major substrates. Furthermore, upon polarization of epithelial cells, the GnT-V products accumulate to the apical side, suggesting that polarized subcellular trafficking is involved in selective modification. Our findings provide insights into the mechanisms of protein-selective glycosylation in vivo.
Keywords:
Biochemistry; Cell biology; Proteomics.