|Human lysyl hydroxylase isoforms: Multifunctionality of human LH3 and the amino acids important for its collagen glycosyltransferase activities|
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Hydroxylysine is found only in animal proteins and mostly in collagens. The hydroxyl groups of hydroxylysine residues have two important functions: they serve as attachment sites for carbohydrates and they play a crucial role in stabilizing intra- and intermolecular crosslinks. Hydroxylysine also exists in collagen-like domains of several other proteins that are not defined as collagens. (Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998).
The content of hydroxylysine in the triple-helical domains of collagen peptide chains varies from 6 [α1(III)] to 67 [α2(VI)] residues per 1,000 amino acids. Only 17% of the lysyl residues are hydroxylated in collagen type III, whereas almost 90% are hydroxylated in collagen type IV and about 80% in collagen type VI. Additional variations are found in hydroxylysine content within the same collagen type and even in the same tissue under different physiological and pathological conditions (Kivirikko & Myllylä 1980, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998).
Hydroxylysine is typically found in triple-helical regions almost exclusively in the Y positions of the repeating -X-Y-Gly- sequences in various collagens. In some collagens it is found from the short nontriple-helical regions at the ends of the α chain as -X-Hyl-Ala- or -X-Hyl-Ser- (Kivirikko & Myllylä 1980, Kivirikko et al. 1992).
The hydroxylation of many lysyl residues in the Y position is not complete and many of them are not hydroxylated at all in some collagens. This explains the differences observed in hydroxylysine content within the same collagen type from various sources, and also partly explains the differences found among various collagen types. The latter may also be due to the differences in total amounts of lysyl residues incorporated per 1,000 amino acids and in the distribution of incorporated lysyl residues between the X and Y positions (Kivirikko & Myllylä 1980, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998).
It seems evident that lysyl residues are more readily hydroxylated in certain triplets than in others. It has been reported that embryonic tissues contain much more hydroxylysine than adult tissues (Kielty et al. 1993, Bateman et al. 1996). There is also evidence that hydroxylation of lysyl residues in collagens is associated with the mineralization of fibrils, the high hydroxylation level preventing a deposit of minerals between fibrils (Wassen et al. 2000). Lysine hydroxylation seems to be increased as well in some diseases, for example, lipodermatosclerosis (Brinckmann et al. 1999), osteoporosis (Köwits et al. 1997, Lo Cascio et al. 1999), and osteogenesis imperfecta (Brenner et al. 1989, Tenni et al. 1993, Lehmann et al. 1995, Bank et al. 2000).
Hydroxylysine has also been identified in some ‘noncollagen’ collagens, such as the C1q subcomponent of complement, acetylcholinesterase, mannose binding proteins, type I and II macrophage scavenger receptors and bovine conglutinin (Kivirikko et al. 1992). Angerfish somatostatin-28 contains a single hydroxylysyl residue in the sequence -Trp-Hyl-Gly- (Andrews et al. 1984, Spiess & Noe 1985). It has been reported that the full-length adiponectin produced from mammalian cells acts functionally as an insulin sensitizer. The four lysyl residues conserved in all species within the collagenous domain are all hydroxylated and sequentially glycosylated. It is suggested that the hydroxylation and glycosylation might contribute to the insulin-sensitizing activity of adiponectin with respect to the inhibition of hepatic glucose production (Wang et al. 2002).
The glycosylation of hydroxylysyl residues occurs as a post-translational event in collagen biosynthesis. The carbohydrates linked to hydroxylysyl residues are either a monosaccharide galactose or a disaccharide glucosylgalactose. The formation of hydroxylysine linked carbohydrate units is catalyzed by two specific enzymes called hydroxylysyl galactosyltransferase (EC 18.104.22.168, GT) and galactosylhydroxylysyl glucosyltransferase (EC 22.214.171.124, GGT) (details see 2.4). The extent of glycosylation of the hydroxylysyl residues and the ratio of galactosylhydroxylysine to glucosylgalactosylhydroxylysine vary considerably among different collagen types and within the same collagen type from various sources (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1984, Kivirikko et al. 1992). Hydroxylysyl residues in the Y position of -Gly-X-Y- triplets in collagen type VI are fully glycosylated (Ayad et al. 1998). The chains of collagen type II and IV are also highly glycosylated from 6% in α1(II) to almost 15% in α1(IV) whereas the chains of collagen type I and III contain very low levels of hydroxylysyl-linked carbohydrate, being only 0.5-1% (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1984). The function of the hydroxylysyl-linked carbohydrate units remains poorly understood. It has been suggested that they may play a role in the control of the organization of the fibrils. Studies on fibrillar collagens have indicated that an increase content of glycosylated hydroxylysyl residues is associated with a decrease of fibril diameter both in vivo and in vitro (Brinckmann et al. 1999, Notbohm et al. 1999). Adiponectin, the fat-derived hormone, has recently been shown to be able to decrease hyperglycemia and to reverse insulin resistance (Berg et al. 2001, Fruebis et al. 2001, Yamauchi et al. 2001). The endogenous adiponectin secreted by adipocytes is post-translationally modified to eight different isoforms and, six of them are glycosylated. The glycosides linked to the four conserved hydroxylysyl residues in the collagenous domain are probably the glucosylgalactosyl group. In vitro mutagenesis data has demonstrated that hydroxylation and glycosylation of the conserved hydroxylysyl residues are required for its insulin-sensitizing action (Wang et al. 2002).
Crosslinking takes place in at least two stages. The initial reaction catalyzed by peptidyl lysyl oxidase is oxidative deamination of ε-amino groups of specific lysyl and hydroxylysyl residues in telopeptide regions of collagens to form reactive aldehydes (allysine and hydroxyallysine). These aldehydes subsequently participate in the formation of the various crosslinks, either by aldol condensation of two of the aldehydes or by condensation between one aldehyde and one ε-amino group of a second lysyl, hydroxylysyl, or glycosylated hydroxylysyl residue. The crosslinks formed from hydroxylysine-derived aldehyde are more stable than those formed from lysine-derived aldehyde (Kivirikko & Myllylä 1984). The lysine aldehyde pathway occurs primarily in adult skin, cornea, and sclera whereas the hydroxylysine aldehyde pathway predominates in bone, cartilage, ligament, most tendons, embryonic skin, and most major internal connective tissues of the body (Eyre 1987, Kielty et al. 1993).