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

Abstract

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. SDS͞PAGE 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. L ysyl hydroxylase (LH, EC 1.14.11.4, Plod) catalyzes the hydroxylation of lysine residues in -X-Lys-Gly-triplets in collagens and other proteins with collagen-like sequences (1). The hydroxylysine residues formed have two important functions: (i) they serve as attachment sites for carbohydrates, either monosaccharide galactose or disaccharide glucosylgalactose, and (ii) they have an important role in the stabilization of intermolecular collagen crosslinks that provide tensile strength and mechanical stability for the collagen fibrils. The extent of lysine hydroxylation and hydroxylysine glycosylation varies between collagen types. Nearly 90% of all lysines are hydroxylated and hydroxylysines glycosylated in types IV and VI collagen, whereas Ͻ20% of the lysines are hydroxylated in type III collagen (2). The functions of the hydroxylysine-linked carbohydrates are poorly understood, but in the case of fibril-forming collagens, they are thought to regulate the formation and morphology of the fibrils (1, 3).Three human LH isoenzymes have been identified (1, 4-6). Mutations in the LH1 gene lead to the kyphoscoliotic type of Ehlers-Danlos syndrome, which is characterized by scoliosis, joint laxity, skin fragility, ocular manifestations, and severe muscle hypotonia (7-9). Mutations in the LH2 gene have recently been reported in two families with Bruck syndrome, which is characterized by fragile bones, joint contractures, scoliosis, and osteoporosis (10).No mutations have been characterized in the gene for the isoenzyme LH3, and its in vivo functions have not yet been elucidated. This protein differs from the other two lysyl hydroxylase isoenzymes in that it also possesses relatively low levels...

Post-translational Modifications of the Four Conserved Lysine Residues within the Collagenous Domain of Adiponectin Are Required for the Formation of Its High Molecular Weight Oligomeric Complex

“…6). Consistent with our results, a recent study on lysyl hydroxylase-3-null mice showed that ablation of this enzyme leads to a total absence of hydroxylysine and its attached carbohydrates in type IV collagen and impaired high order structure formation of this protein (43). Notably, in addition to its lysyl hydroxylase activity, lysyl hydroxylase-3 also possesses relatively low levels of glucosyltransferase and galactosyltransferase activities, suggesting that this enzyme alone is sufficient for catalyzing all three steps, including lysine hydroxylation and its further attachment with an ␣-1,2-glucosylgalactosyl group (45).…”

“…Minoxidil has been shown to inhibit the activity of lysyl hydroxylase by decreasing the gene expression of all three isoenzymes (42,43). We therefore tested the effect of minoxidil on the oligomeric complex formation of endogenous adiponectin in primary rat adipocytes.…”

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

“…While the tissue distribution of lh gene expression has been examined by Northern blot analysis, with the exception of lh3, there have been no published accounts of the in situ expression patterns of these genes during embryogenesis in any vertebrate species (Passoja et al, 1998b;Rautavuoma et al, 2004;Ruotsalainen et al, 1999;Valtavaara et al, 1997;Valtavaara et al, 1998;Yeowell et al, 1994;Yeowell and Walker, 1999). In this paper, we report the cloning of zebrafish lh1 and lh2 and their patterns of expression throughout the first two days of development.…”

“…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). More recently, we have demonstrated that diwanka, the zebrafish homolog of lh3, plays a specific role in motor axon migration (Schneider and Granato, 2006).…”

Development of a convenient peptide‐based assay for lysyl hydroxylase

“…17,18 LH3 null mice have defective type IV collagen biosynthesis, leading to embryonic lethality. 19 Thus, studies of LH isoforms are of interest not only for their physiological functions but also for their roles in inheritable human diseases.…”