Abstract
Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.
Highlights
Collagens comprise a large protein family that is ubiquitously distributed throughout the body
Analysis of a hypomorphic Lysyl hydroxylase 3 (LH3) mouse line with the same mutation, demonstrated that the reduction of the galactosylhydroxylysyl glucosyltransferase (GGT) activity of LH3 disrupts the localization of type IV collagen, and the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5
The results confirm that LH3 has lysyl hydroxylase (LH) and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane
Summary
Collagens comprise a large protein family that is ubiquitously distributed throughout the body. Collagen biosynthesis involves many post-translational modifications some of which are unique to collagens and proteins with collagenous domains (Bateman et al, 1996; Kielty and Grant, 2002; Kivirikko et al, 1992; Myllyharju and Kivirikko, 2001) These include the hydroxylation of lysine residues, and glycosylation of hydroxylysine residues to galactosylhydroxylysine and glucosylgalactosylhydroxylysine residues (Kivirikko and Myllylä, 1979). The modified amino acids, located in the Y position of the repeating X-Y-Gly triplets in the triple-helical region of collagens, extend outward from the helix and form the surface of the molecules (Kielty and Grant, 2002) These modifications are probably important for protein-protein and protein-cell interactions. Recent studies suggest that in adiponectin, the hydroxylation and glycosylation of lysine residues in the collagenous domain contribute to the insulin-sensitizing activity of the hormone (Wang, Y. et al, 2002)
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