Characterization of Three Fragments That Constitute the Monomers of the Human Lysyl Hydroxylase Isoenzymes 1–3

Abstract

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80 -85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä , R. (2000) J. Biol. Chem. 275, 36158 -36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

“…In addition to LH activity, LH3, but not LH1 or LH2, also possesses collagen glucosyltransferase activity and very small amounts of collagen galactosyltransferase activity (11)(12)(13), implying that this protein may be able to perform all of the reactions involved in the formation of hydroxylysine and its carbohydrate units. The level of collagen galactosyltransferase activity was not decreased in the LH3 null embryos, but collagen glucosyltransferase activity had decreased to Ϸ15% in the null embryos, indicating that this activity of LH3 is of real biological significance and may contribute to the phenotype in null mice.…”

“…This protein differs from the other two lysyl hydroxylase isoenzymes in that it also possesses relatively low levels of collagen glucosyltransferase activity and very small amounts of collagen galactosyltransferase activity in addition to the lysyl hydroxylase activity (11)(12)(13). In this study, we generated mice with targeted inactivation of the LH3 (Plod3) gene and show that LH3 is essential for the synthesis of type IV collagen during early development and, hence, for the stability of basement membranes (BMs).…”

Development of a convenient peptide‐based assay for lysyl hydroxylase

“…10,11,14 Peptides 1-4 were obtained in high purity, as indicated by homogenous RP-HPLC elution profiles (data not shown) and MALDI-TOF MS values (see Materials and Methods). The CD spectrum for peptide 1 (Figure 2) was virtually identical to that previously reported.…”

“…9 LH3 differs from LH1 and LH2 in that it is multifunctional and able to catalyze, in addition to Lys hydroxylation, sugar transfer reactions, the subsequent steps in the formation of glucosylgalactosyl-Hyl residues. [12][13][14] Only one isoform for LH is present in lower species such as Caenorhabditis elegans. 9,15 This ancestral C. elegans LH is also able to glycosylate Hyl residues and, thus, is functionally similar to LH3.…”

Genomic structure and embryonic expression of zebrafish lysyl hydroxylase 1 and lysyl hydroxylase 2

“…Recently, mutations in human lh2 (plod2) were linked to Bruck syndrome, distinguished by fragile bones and contractures of the large joints (Ha-Vinh et al, 2004;van der Slot et al, 2003). LH3, in addition to exhibiting lysyl hydroxylase activity, is the only LH enzyme to possess additional glycosyltransferase activities that serve to further modify hydroxylysine residues to galactosylhydroxylysine and glucosylgalactosylhydroxylysine (Heikkinen et al, 2000;Rautavuoma et al, 2002;Wang et al, 2002). Although mutations in human lh3 (plod3) have not yet been identified, a mouse knockout reveals this gene has an embryonic lethal phenotype and is required for type IV collagen secretion (Rautavuoma et al, 2004;Ruotsalainen et al, 2006).…”