The Muscle-Specific Kinase (MuSK) is a monotopic transmembrane receptor responsible for key signaling events during development and maintenance of neu…

Researchers comment on two recent Nature Communications publications on enzyme complexes involved in collagen glycosylation, discussing how these findings lay a foundation for understanding collagen I...

Collagen IV, an essential and evolutionarily conserved component of basement membranes, is one of the most extensively post-translationally modified p…

Hydroxylysine galactosylation is essential for collagen maturation. This work reveals the molecular structure of human GLT25D1, its catalytic residues, and a noncatalytic structural domain stabilized ...

In una manifestazione promossa da Coldiretti, principale organizzazione degli imprenditori agricoli italiani, che si è svolta mercoledì 19 marzo a Parma, si sono radunate, secondo gli organizzatori, 2...

Si tratta dei progetti di ricerca di Riccardo Piccinno e di Giovanni Stelitano, che studieranno rispettivamente un batterio che vive ospite nel moscerino dei piccoli frutti e lo sviluppo di nuovi antimicrobici per combattere patogeni opportunisti.

During biosynthesis, collagen lysine residues undergo extensive post-translational modifications essential for the stability and functions of collagen supramolecular assemblies. In the endoplasmic reticulum, two distinct metal ion dependent enzyme families (i.e., multifunctional lysyl hydroxylases-glucosyltransferases LH/PLODs and galactosyltransferases GLT25D/COLGALT) alternatively operate on collagen lysine side chains ultimately generating the highly conserved α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in the collagen lysine post-translational modification machinery is linked to multiple developmental pathologies as well as extracellular matrix alterations causing enhanced cellular proliferation and invasiveness of several solid tumors, prompting for an in-depth characterization of LH/PLOD and GLT25D/COLGALT enzyme families. Here, we present an integrative molecular study of GLT25D1/COLGALT, highlighting an elongated head-to-head homodimeric assembly characterized by an N-terminal segment of each monomer wrapping around its dimerization partner. Each monomer encompasses two Rossman fold-type domains (GT1 and GT2) separated by an extended linker. Both domains were found capable of binding Mn2+ cofactors and UDP-α-galactose donor substrates, resulting in four candidate catalytic sites per dimer. Site-directed mutagenesis and biochemical studies identify the C-terminal GT2 domain as the functional GLT25D1/COLGALT1 catalytic site, highlighting an unprecedented Glu-Asp-Asp motif critical for metal ion binding, and suggesting structural roles for the N-terminal GT1 essential for correct quaternary structure assembly. Conversely, dimerization was not a requirement for GLT25D1/COLGALT1 enzymatic activity in vitro , suggesting that the elongated enzyme homodimer assembly, resembling that of LH/PLOD binding partners, could represent a functional hallmark for correct recognition and successful processing of collagen lysine residues. ### Competing Interest Statement The authors have declared no competing interest.

20 June 2024- 2pm BST (9am NY / 10am Canada / 3pm CEST)- on Zoom Title: Greening your lab; Sustainable Practices & Benefits for your Life Science Lab Speakers: Julie Di Martino, New York Medical College, USA & Federico Forneris, University of Pavia, Italy

Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the glycosylation of collagen lysines, although a precise description of the contribution of each enzyme to these essential PTMs has not yet been provided in the literature. LH3/PLOD3 is thought to be capable of performing two chemically distinct collagen glycosyltransferase reactions using the same catalytic site: an inverting beta-1,O-galactosylation of hydroxylysines (Gal-T) and a retaining alpha-1,2-glucosylation of galactosyl hydroxylysines (Glc-T). In this work, we have combined indirect luminescence-based assays with direct mass spectrometry-based assays and molecular structure studies to demonstrate that LH3/PLOD3 only has Glc-T activity and that GLT25D1 only has Gal-T activity. Structure-guided mutagenesis confirmed that the Glc-T activity is defined by key residues in the first-shell environment of the glycosyltransferase catalytic site as well as by long-range contributions from residues within the same glycosyltransferase (GT) domain. By solving the molecular structures and characterizing the interactions and solving the molecular structures of human LH3/PLOD3 in complex with different UDP-sugar analogs, we show how these studies could provide insights for LH3/PLOD3 glycosyltransferase inhibitor development. Collectively, our data provide new tools for the direct investigation of collagen hydroxylysine PTMs and a comprehensive overview of the complex network of shapes, charges, and interactions that enable LH3/PLOD3 glycosyltransferase activities, expanding the molecular framework and facilitating an improved understanding and manipulation of glycosyltransferase functions in biomedical applications.