Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

Funding Information: This work was supported by Fundação para a Ciência e a Tecnologia, I. P. through iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (to J.C. and J.B.V.) and by US NIH Grants GM130915, GM103390, and GM107012 (to K.W.M.). Funding Information: Mass spectrometry data were provided/obtained by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. We thank the technical assistance of Conceição Almeida, UniMS and Cristina Leitão, ITQB NOVA with the MS and HPAEC-PAD analyses, respectively. We thank Dr. Pedro Lamosa, CERMAX, ITQB NOVA, for the NMR analysis. iBET is acknowledged for access to ITF-DSF equipment. Publisher Copyright: © 2023, The Author(s). ; Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes. ; publishersversion ; published