Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.