Secretion and Assembly of Type IV and VI Collagens Depend on Glycosylation of Hydroxylysines

Abstract

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. Collagen biosynthesis contains many co-and post-translational modifications that are unique to collagenous proteins. These modifications include hydroxylation of lysyl residues and their further glycosylation to galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues. Lysyl hydroxylase (LH 2 ; E.C. 1.14.11.4) catalyzes the hydroxylation of lysyl residues in the Y-position of the repeating collagen Gly-X-Y triplets (1, 2), and three lysyl hydroxylase isoforms have been characterized: LH1, LH2, and LH3 (3-10).LH3 is a multifunctional enzyme containing, in addition to lysyl hydroxylase activity, collagen galactosyltransferase (E.C. 2.4.1.50) and glucosyltransferase (E.C. 2.4.1.66) activities in vitro (11-13). LH3 is functional in both the endoplasmic reticulum and extracellular space (14), and loss of LH3 leads to early embryonic lethality (15, 16). We have reported recently that LH3 is also multifunctional in vivo and that especially the glucosyltransferase activity of LH3 is essential for normal embryonic development (16).The amount of lysine hydroxylation and glycosylation varies between different collagen types. Type IV and VI collagens are highly hydroxylated and glycosylated, whereas fibrillar collagens, especially types I and III, have much lower levels of these modifications (1, 17, 18). The hydroxylysyl residues have an important role in collagen cross-linking (19,20), but the function of the glycosylation of the hydroxylysyl residues is still poorly understood. The role of glycosylated hydroxylysines in the formation and morphology of collagen fibrils has been debated (21, 22), and we have shown that they are necessary for the correct basement membrane localization of type IV collagen (16).We produced previously three different mouse lines with a manipulated LH3 gene (16). The mice with no or a very low level of the LH3 protein, thus lacking the lysyl hydroxylase and glucosyltransferase activities of LH3, had an embryonic lethal phenotype. The mouse line (LH mutant) with a point mutation that specifically destroys the LH activity of LH3 without affecting glycosyltransferase activities (11) was viable, but showed ultrastructural alterations (16). In this study, we used LH3 knock-out and LH mutant mice and fibroblasts derived from them as a model to investigate the functions of glycosylated hydroxylysines of collagens. This work provides new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion of type IV collagen and in the tetramerization and *

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…Interestingly, COL1 residue 84 was detected here as both a Glc-Gal-Hyl and a Gal-Hyl residue, showing glycosylation at this site to be dynamic, with partial occupancy by both Glc-Gal and Gal saccharides. Our direct localization of 30 COL1 Hyl residues within the human recombinant pro-␣1(V) supersedes a previous amino acid analysis estimate of 6 COL1 Hyl residues for human recombinant pro-␣1(V) chains produced in 293-HEK cells (61) and is more congruent with the observed efficient secretion of recombinant pro-␣1(V) 3 homotrimers from 293-HEK cells (40,61), as threshold levels of glycosylated Hyl residues are necessary for efficient secretion of at least some collagenous molecules (18).…”

“…Functionally, 4-Hyp residues have well characterized effects in stabilizing triple helices (16), whereas Hyl residues are important to the formation of stable covalent cross-links between collagen chains (17), and Hyl glycosylation may be important in assembly and secretion of at least some collagens (18). The function of 3-Hyp residues is unclear, with conflicting reports on small stabilizing (19) or destabilizing (20) effects on triple helix stability.…”

“…Thus, 29 hydroxylated Lys residues found in ␣1(V) COL1 domains from two very different sources may be relatively invariant in ␣1(V) chains. The reduced numbers of Hyl and Glc-Gal-Hyl residues in the human recombinant protein suggests that 293-HEK cells, which produce little or no endogenous extracellular matrix proteins (61), may possess reduced levels of the multifunctional enzyme lysyl hydroxylase 3, which has lysyl hydroxylase activity, and collagen galactosyltransferase and glycosyltransferase activities that allow it to glycosylate Hyl residues hydroxylated by itself and by other lysl hydroxylase isoforms (18), although even normal levels of enzymes might be insufficient to fully modify the overexpressed recombinant pro-␣1(V)chains. Interestingly, COL1 residue 84 was detected here as both a Glc-Gal-Hyl and a Gal-Hyl residue, showing glycosylation at this site to be dynamic, with partial occupancy by both Glc-Gal and Gal saccharides.…”

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

“…4 indicate increased glycosylation of collagen VI in heart failure (due to the greater increase in WGA binding relative to the smaller increase in the amount of collagen VI protein). Importantly, glycosylation of hydroxylysines is essential for the function of collagen VI (Sipilä et al 2007). Although the significance of this increased WGA binding is not presently known, if it was linked to ttubule remodelling, it could potentially be targeted.…”

Missense Mutations That Cause Bruck Syndrome Affect Enzymatic Activity, Folding, and Oligomerization of Lysyl Hydroxylase 2

“…Interestingly, the lethal phenotype of LH3 null embryos is caused by a lack of the glycosyltransferase activity of LH3 and not of its LH activity (21). This glycosyltransferase activity has been shown to be important for the proper assembly and secretion of collagens IV and VI (22) and for cell growth and viability (23). A patient with a human connective tissue disorder caused by mutations in the gene for LH3 was reported very recently to be a compound heterozygote with mutations leading to a marked reduction in the glycosyltransferase and LH activities of LH3 and a decrease in the amount of LH3 polypeptide (24).…”