Delving into human α1,4-galactosyltransferase acceptor specificity: the role of enzyme dimerization

Abstract

Glycosyltransferases (GTs) exhibit precise donor and acceptor specificities, governed by intricate mechanisms, including protein assembly. Human α1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, catalyzes the synthesis of Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), receptors for Shiga toxin type 1 (Stx1) and 2 (Stx2). These toxins are produced by enterohemorrhagic Escherichia coli and can lead to severe outcomes, such as hemolytic-uremic syndrome (HUS). To elucidate the molecular basis of A4galt specificity towards GSLs and GPs, we explored its interaction with GP-specific β1,4-galactosyltransferase 1 (B4galt1) and GSL-specific β1,4-galactosyltransferase isoenzymes 5 and 6 (B4galt5 and B4galt6). Using a novel NanoBiT assay, we demonstrated that A4galt could form homodimers and heterodimers with B4galt1 and B4galt5. Furthermore, by employing AlphaFold for state-of-the-art structural prediction, we analyzed the interactions and structures of these enzyme complexes. Our analysis highlighted that the A4galt-B4galt5 heterodimer exhibited the highest prediction confidence, indicating a significant role of A4galt heterodimers in determining enzyme specificity toward GSLs and GPs. These insights not only increase our knowledge about glycosylation processes but also open avenues for developing new therapeutic approaches to Shiga toxin-related diseases.