|Human lysyl hydroxylases: Characterization of a novel isoenzyme and its gene, determination of the domain structure of the lysyl hydroxylase polypeptides and generation of knock-out mice for the novel isoenzyme|
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Hydroxylysine is found in vertebrate proteins mostly in the collagens and collagen-like domains of other proteins that are not defined as collagens. The hydroxyl groups of hydroxylysine residues have two important functions: they serve as attachment sites for carbohydrate units and they are crucial for the stability of the intramolecular and intermolecular collagen crosslinks (Kivirikko & Pihlajaniemi 1998).
Hydroxylysine is almost exclusively found in the Y positions of the repeating Gly-X-Y sequences in collagens and proteins with collagen-like domains. An exception to this rule is found in some fibril-forming collagens, where the short non-triple-helical regions at the ends of the α chain contain the sequences X-Hyl-Ser and X-Hyl-Ala (Kivirikko et al. 1992).
The amount of hydroxylysine varies greatly between collagen types, and also within the same collagen type from different sources and even in the same tissue in different physiological and pathological conditions (Kivirikko & Myllylä 1980, Kivirikko et al. 1992). This is at least partly explained by incomplete hydroxylation of many lysine residues in the Y position, and in some collagens a number of lysine residues in these positions are not hydroxylated at all (Kivirikko & Pihlajaniemi 1998).
The hydroxylation of lysine residues also seems to be age-related, so that the hydroxylysine content of collagen from embryonic tissues is higher than of that of collagen from adult tissues (Bailey & Shimokomaki 1971). The amount of hydroxylysine is also increased in some diseases, e.g. osteoporosis (Bailey et al. 1992, Knott et al. 1995, Lo Cascio et al. 1999), osteogenesis imperfecta (Lehmann et al. 1995a, Bank et al. 2000), osteosarcoma (Shapiro & Eyre 1982, Lehmann et al. 1995b) and lipodermatosclerosis (Shapiro & Eyre 1982, Lehmann et al. 1995b, Brinckmann et al. 1999).
Hydroxylysine is also found in most, if not all, proteins with collagenous sequences not defined as collagens (Kielty & Grant 2002, Myllyharju & Kivirikko 2003). It has recently been shown that the fat-derived-hormone adiponectin has the ability to reduce hyperglycaemia and to reserve insulin resistance, and that hydroxylation and subsequent glycosylation of the four lysines in the collagenous domain of this protein may contribute to this activity (Y. Wang et al. 2002).
In addition to proteins with collagenous sequences, hydroxylysine residues are found in some proteins without such sequences. One example is anglerfish somatostatin-28 which is a peptide hormone consisting of 28 amino acids, residue 23 being hydroxylysine in the sequence Trp-Hyl-Gly. The function of this hydroxylysine remains uncertain (Andrews et al. 1984).
Hydroxylysine residues have at least two important functions in collagens: they serve as attachment sites for carbohydrates and they play a crucial role in stabilizing intramolecular and intermolecular crosslinks (Kivirikko & Pihlajaniemi 1998).
The carbohydrates linked to hydroxylysine residues are either the monosaccharide galactose or the disaccharide glucosylgalactose. Formation of these carbohydrate moieties involves two specific enzymes, a galactosyltransferase (E.C. 184.108.40.206) and a glucosyltransferase (E.C. 220.127.116.11). The extent of glycosylation of hydroxylysine residues varies greatly between collagen types, and even within the same collagen type in various physiological and pathological states (Kivirikko & Myllylä 1979, 1980, Kivirikko et al. 1992).
The functions of the hyroxylysine-linked carbohydrate units are not fully understood, but it has been suggested that they may regulate the packing of collagen molecules into supramolecular assemblies, as they are situated on the surface of the collagen molecules (Kivirikko & Pihlajaniemi 1998). Studies of fibrillar collagens have indicated that collagens containing high amounts of hydroxylysine and hydroxylysine-linked carbohydrates form very thin fibrils, whereas collagens with low amounts of these modifications form thick fibrils (Notbohm et al. 1999). As mentioned above, hydroxylation and glycosylation of the four lysines in the collagenous domain of adiponectin may contribute to the insulin-sensitizing ability of this protein (Y. Wang et al. 2002).
The formation of covalent intramolecular and intermolecular crosslinks is the final step in collagen biosynthesis that occurs as an extracellular process after cleavage of the propeptides and fibril assembly (Kadler et al. 1996). These crosslinks are essential in providing the tensile strength and mechanical stability of the collagen fibrils and other supramolecular assemblies (Hulmes 1992). Two crosslinking pathways can be distinguished for fibrillar collagens (Kielty & Grant 2002): the lysine aldehyde pathway , which occurs mainly in adult skin, cornea and sclera, and the hydroxylysine aldehyde pathway, which occurs mainly in the bone, cartilage, ligament, tendons, embryonic skin and most internal organs (Eyre 1987, Kielty & Grant 2002). Crosslinks formed in the hydroxylysine-derived aldehyde pathway are more stable than those formed in the lysine-derived pathway (Kielty & Grant 2002).
Crosslinking of collagens is initiated by the enzyme lysyl oxidase (Smith-Mungo & Kagan 1998, Csiszar 2001, Kagan & Li 2003), which catalyzes oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues situated in the telopeptide regions of collagens to form reactive aldehydes (allysine and hydroxyallysine). These aldehydes then form crosslinks, either by aldol condensation between two of the aldehydes or by condensation between one aldehyde and one ε-amino group of an unmodified lysine, hydroxylysine or glycosylated hydroxylysine residue (Eyre 1987, Bailey et al. 1998, Kielty & Grant 2002).