Lysyl hydroxylase (EC 1.14.11.4) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.Collagens are extracellular proteins found essentially in all tissues. They play a crucial role in maintenance of the structural integrity of tissues and in regulation of cellular behavior. The collagens, like other extracellular proteins, bind to growth factors and other regulatory components of cells and modulate cellular metabolism. To date, 19 genetically distinct collagen types have been identified (1-3). The collagen molecule is composed of three polypeptide chains, which coil around each other into a triple helical structure. Some of the collagen types, such as type I, II, and III collagens, have a rod-like structure without any interruptions in the helical region, whereas in the other types the triple-helical regions are interrupted with multiple short nonhelical sequences. Collagen molecules are aggregated in tissues into supramolecular structures such as fibrils, beaded filaments, or net-like or other kinds of structures depending on the type of collagen (3).The biosynthesis of collagen involves several post-translational modifications, which include hydroxylation of lysyl residues, galactosylation of hydroxylysyl residues, and glucosylation of galactosylhydroxylysyl residues. These reactions occur in the endoplasmic reticulum before triple helix formation. Hydroxylysine occurs in the Y position of the repeating XaaYaa-Gly triplet within the helical region of collagen molecules and also in the sequence of the nonhelical telopeptide regions of some collagen types when glycine is replaced either by serine or alanine (2, 4). The hydroxy groups of hydroxylysyl residues provide attachment sites for glycosyl residues, either the monosaccharide galactose or the disaccharide glucosylgalactose.The hydroxy groups also play a crucial role in the formation of inter-and intramolecular collagen cross-links. The biological role of the hydroxylysyl-linked carbohydrates, which are collagen-specific structures, is not clear. These carbohydrates point outward from the collagen helix and, thus, are located at the surface of the protein where they probably play an important role in lateral interactions between collagen triple helices and between collagen molecules and other extracellular ...
Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling. Hydroxylysine and its glycosylations are post-translational modifications involved in collagen biosynthesis. There are several comprehensive reviews, which discuss collagen biosynthesis and diseases linked to collagens (reviewed in Kielty et al., 1993;Kadler, 1994;Bateman et al., 1996; Kivirikko, 2001, 2004). The focus of the current review is to update the recent discovery that LH3 has multiple activities in the modification of lysyl residues in vivo, the importance of the various activities of LH3 during embryonic development, and the localization of functional LH3 both in the ER and in the extracellular space. The hydroxylation of lysine is one of the modifications (reviewed in Kivirikko et al., 1992;Kielty et al., 1993;Kadler, 1994;Prockop and Kivirikko, 1995;Bateman et al., 1996;Myllyharju and Kivirikko, 2001), which is characteristic of collagens. Hydroxylysyl residues in fibrillar collagens participate in the formation of collagen cross-links that connect molecules to each other and stabilize the extracellular matrix. The hydroxylysyl residues within the telopeptide region of the collagen molecule are converted to hydroxyallysines, which subsequently react with lysyl or hydroxylysyl residues within the triple helix to form deoxypyridinoline and pyridinoline cross-links, respectively (Robins and Brady, 2002;Eyre and Wu, 2005...
Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.
Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllylä , R. (2000) J. Biol. Chem. 275, 36158 -36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo.
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