Selective modification of glycoprotein substrates by GnT-V in mouse kidney

<i>N</i>-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 <i>N</i>-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 <i>Mgat5</i> and <i>Anpep</i> or <i>Mep1a</i>, excluding the possibility that <i>Mgat5</i> 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 <i>in vivo</i>.