Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.

Lysyl hydroxylase (LH), with three isoenzymes in vertebrates, catalyzes the formation of hydroxylysine by acting on -X-Lys-Gly- triplets in the collagenous domains of proteins of the collagen superfamily and also in -X-Lys-Ala- or -X-Lys-Ser- sequences in the telopeptides located at the ends of the polypeptide chains in some fibril-forming collagens. The hydroxylysine residues are essential for the stability of collagen crosslinks and act as carbohydrate attachment sites. The extent of lysine hydroxylation varies between collagen types, between tissues in the same collagen type and in certain diseases, suggesting that the LH isoenzymes may have different substrate specificities. We studied here the hydroxylation of synthetic peptides representing various hydroxylation sites in type I and IV collagens by purified recombinant LHs in vitro and of a recombinant full-length type I procollagen chain coexpressed with each LH in insect cells. All three LHs hydroxylated peptides representing collagenous sequences of type I and IV collagens, although with different K(m) and V(max) values. Furthermore, all three hydroxylated the collagenous domain of the coexpressed type I procollagen chain to a similar extent. None of the isoenzymes hydroxylated peptides representing the N and C telopeptides of type I collagen, but LH2, unlike the other two isoenzymes, hydroxylated the N telopeptide in the coexpressed procollagen chain. Hydroxylation of the telopeptide lysines by LH2 thus occurs only in the context of a long peptide. These data provide the first direct evidence that LH2 is a specific telopeptide hydroxylase, while all three LHs act on collagenous sequences.

Tissue-specific changes in the hydroxylysine content and cross-links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock-out mice.

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

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

Collagens carry hydroxylysine residues that act as attachment sites for carbohydrate units and are important for the stability of crosslinks but have been regarded as nonessential for vertebrate survival. We generated mice with targeted inactivation of the gene for one of the three lysyl hydroxylase isoenzymes, LH3. The null embryos developed seemingly normally until embryonic day 8.5, but development was then retarded, with death around embryonic day 9.5. Electron microscopy (EM) revealed fragmentation of basement membranes (BMs), and immuno-EM detected type IV collagen within the dilated endoplasmic reticulum and in extracellular aggregates, but the typical BM staining was absent. Amorphous intracellular and extracellular particles were also seen by collagen IV immunofluorescence. SDS/PAGE analysis demonstrated increased mobilities of the type IV collagen chains, consistent with the absence of hydroxylysine residues and carbohydrates linked to them. These results demonstrate that LH3 is indispensable for biosynthesis of type IV collagen and for BM stability during early development and that loss of LH3's functions leads to embryonic lethality. We propose that the premature aggregation of collagen IV is due to the absence of the hydroxylysine-linked carbohydrates, which thus play an essential role in its supramolecular assembly.

p38 Mitogen-activated protein kinase regulates interleukin-4-induced gene expression by stimulating STAT6-mediated transcription.

STAT6 functions as a critical mediator of IL-4-stimulated gene activation, and the function of STAT6 is regulated by both tyrosine and serine kinase activities. Here we analyzed the role of serine phosphorylation in regulation of STAT6-mediated transcription. Optimal transcriptional response of IL-4-inducible promoters requires costimulatory signals through CD40-stimulated intracellular kinases such as p38 MAPK. We found that the p38 MAPK inhibitor SB202190 as well as the dominant negative p38 MAPK inhibited interleukin (IL)-4 regulated expression of CD23 in Ramos B cells. IL-4 stimulation did not stimulate p38 MAPK activity, but inhibition of p38 MAPK activity directly correlated with inhibition of IL-4-induced gene activation. Dissection of individual response elements on IL-4-regulated promoter showed that C/EBP beta-mediated transcription was insensitive to SB202190 treatment in B cells whereas STAT6-mediated transcription was regulated by p38 MAPK. The IL-4-induced immediate activation events of STAT6 were not affected by p38 MAPK activity. Furthermore, phosphoamino acid analysis and phosphopeptide mapping indicated that STAT6 is not a direct substrate for p38 MAPK. Instead, p38 MAPK was found to directly regulate the activity of the transactivation domain of STAT6. These results show that, in addition to the well established proinflammatory effects, p38 MAPK also provides a costimulatory signal for IL-4-induced gene responses by directly stimulating the transcriptional activation of STAT6.

Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity.

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.