Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity

Mattoteia, Daiana; Chiapparino, Antonella; Fumagalli, Marco; De Marco, Matteo; De Giorgi, Francesca; Negro, Lisa; Pinnola, Alberta; Faravelli, Silvia; Roscioli, Tony; Scietti, Luigi; Forneris, Federico

doi:10.3390/ijms241311213

Open AccessArticle

Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity

by

Daiana Mattoteia

^1,†,

Antonella Chiapparino

^1,†,

Marco Fumagalli

^1,‡,

Matteo De Marco

^1,‡

,

Francesca De Giorgi

^1,‡

,

Lisa Negro

¹,

Alberta Pinnola

¹,

Silvia Faravelli

¹,

Tony Roscioli

^2,3,

Luigi Scietti

^1,§

and

Federico Forneris

^1,4,*

¹

The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9A, 27100 Pavia, Italy

²

NSW Health Pathology Randwick Genomics Laboratory, Prince of Wales Hospital, Sydney, NSW 2031, Australia

³

Neuroscience Research Australia (NeuRA), Prince of Wales Clinical School, University of New South Wales, Sydney, NSW 2052, Australia

⁴

Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo, 27100 Pavia, Italy

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

^‡

These authors contributed equally to this work.

^§

Present address: Biochemistry and Structural Biology Unit, Department of Experimental Oncology, IRCCS European Institute of Oncology (IEO), Via Adamello 16, 20139 Milan, Italy.

Int. J. Mol. Sci. 2023, 24(13), 11213; https://doi.org/10.3390/ijms241311213

Submission received: 2 April 2023 / Revised: 22 June 2023 / Accepted: 5 July 2023 / Published: 7 July 2023

(This article belongs to the Special Issue Recent Advances in Collagen Proteins)

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Abstract

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.

Keywords: collagen biosynthesis; extracellular matrix; post-translational modifications; collagen glycosylations; glycosyltransferase; lysyl hydroxylase

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MDPI and ACS Style

Mattoteia, D.; Chiapparino, A.; Fumagalli, M.; De Marco, M.; De Giorgi, F.; Negro, L.; Pinnola, A.; Faravelli, S.; Roscioli, T.; Scietti, L.; et al. Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity. Int. J. Mol. Sci. 2023, 24, 11213. https://doi.org/10.3390/ijms241311213

AMA Style

Mattoteia D, Chiapparino A, Fumagalli M, De Marco M, De Giorgi F, Negro L, Pinnola A, Faravelli S, Roscioli T, Scietti L, et al. Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity. International Journal of Molecular Sciences. 2023; 24(13):11213. https://doi.org/10.3390/ijms241311213

Chicago/Turabian Style

Mattoteia, Daiana, Antonella Chiapparino, Marco Fumagalli, Matteo De Marco, Francesca De Giorgi, Lisa Negro, Alberta Pinnola, Silvia Faravelli, Tony Roscioli, Luigi Scietti, and et al. 2023. "Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity" International Journal of Molecular Sciences 24, no. 13: 11213. https://doi.org/10.3390/ijms241311213

APA Style

Mattoteia, D., Chiapparino, A., Fumagalli, M., De Marco, M., De Giorgi, F., Negro, L., Pinnola, A., Faravelli, S., Roscioli, T., Scietti, L., & Forneris, F. (2023). Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity. International Journal of Molecular Sciences, 24(13), 11213. https://doi.org/10.3390/ijms241311213

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Identification of Regulatory Molecular “Hot Spots” for LH/PLOD Collagen Glycosyltransferase Activity

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