Matrix Biology 21 (2002) 559–566 0945-053X/02/$ - see front matter  2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. PII:S0945-053X Ž 02 . 00071-9 The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro Chunguang Wang , Hanne Luosujarvi , Jari Heikkinen , Maija Risteli, Lahja Uitto, Raili Myllyla* 1 1 1 ¨ ¨ Biocenter and the Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland Received 30 May 2002; received in revised form 8 August 2002; accepted 8 August 2002 Abstract Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys and aspartates in 144 positions 187–191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.  2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. Keywords: Galactosyltransferase; Lysyl hydroxylase; Collagen biosynthesis 1. Introduction Collagens are a family of proteins found essentially in all animal tissues. They form a diverse range of highly organized supramolecular assemblies in the extra- cellular matrix and generate structural scaffolds to sup- port cells within tissues, connect tissues within an organ and facilitate attachment and migration of cells. In addition, collagens, like other proteins, bind to growth factors as well as to other regulatory components of cells and modulate cellular metabolism and behavior. Abbreviations: LH, lysyl hydroxylase; GT, hydroxylysyl galacto- syltransferase; GGT, galactosylhydroxylysyl glucosyltransferase; PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl b-D- thiogalactoside. *Corresponding author. Fax: q358-8-5531141. E-mail address: raili.myllyla@oulu.fi (R. Myllyla). ¨ These authors contributed equally. 1 The biosynthesis of collagens involves many post- translational modifications, which include hydroxylation of lysyl residues and glycosylation of hydroxylysyl residues occurring in the endoplasmic reticulum (Kivi- rikko et al., 1992; Kielty et al., 1993; Kadler 1994; Prockop and Kivirikko, 1995; Bateman et al., 1996; Knott and Bailey, 1998). The number of hydroxylated lysyl residues and glycosylated hydroxylysine residues varies among different collagen types but also within the same collagen type in different tissues and in different physiological conditions. Embryonic collagens, for instance, are more extensively modified than adult collagens (above Refs.), and studies on bone collagen indicate that the lysyl modifications vary among differ- ent skeletal regions (Moro et al., 2000). Hydroxylysine has an important function in collagen cross-link forma- tion in vivo, especially in weight-bearing and mineral- ized tissues such as bone and cartilage (Kielty et al.,