Lysyl Hydroxylase 3 Is a Multifunctional Protein Possessing Collagen Glucosyltransferase Activity

Heikkinen, Jari; Risteli, Maija; Wang, Chunguang; Latvala, Jaana; Rossi, Maarit; Valtavaara, Minna; Myllylä, Raili

doi:10.1074/jbc.m006203200

Cited by 126 publications

(143 citation statements)

References 18 publications

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“…In addition to LH activity, LH3, but not LH1 or LH2, also possesses collagen glucosyltransferase activity and very small amounts of collagen galactosyltransferase activity (11)(12)(13), implying that this protein may be able to perform all of the reactions involved in the formation of hydroxylysine and its carbohydrate units. The level of collagen galactosyltransferase activity was not decreased in the LH3 null embryos, but collagen glucosyltransferase activity had decreased to Ϸ15% in the null embryos, indicating that this activity of LH3 is of real biological significance and may contribute to the phenotype in null mice.…”

Section: Discussionmentioning

confidence: 99%

“…This protein differs from the other two lysyl hydroxylase isoenzymes in that it also possesses relatively low levels of collagen glucosyltransferase activity and very small amounts of collagen galactosyltransferase activity in addition to the lysyl hydroxylase activity (11)(12)(13). In this study, we generated mice with targeted inactivation of the LH3 (Plod3) gene and show that LH3 is essential for the synthesis of type IV collagen during early development and, hence, for the stability of basement membranes (BMs).…”

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confidence: 99%

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Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Rautavuoma

Takaluoma

Sormunen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

102

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Collagens carry hydroxylysine residues that act as attachment sites for carbohydrate units and are important for the stability of crosslinks but have been regarded as nonessential for vertebrate survival. We generated mice with targeted inactivation of the gene for one of the three lysyl hydroxylase isoenzymes, LH3. The null embryos developed seemingly normally until embryonic day 8.5, but development was then retarded, with death around embryonic day 9.5. Electron microscopy (EM) revealed fragmentation of basement membranes (BMs), and immuno-EM detected type IV collagen within the dilated endoplasmic reticulum and in extracellular aggregates, but the typical BM staining was absent. Amorphous intracellular and extracellular particles were also seen by collagen IV immunofluorescence. SDS͞PAGE analysis demonstrated increased mobilities of the type IV collagen chains, consistent with the absence of hydroxylysine residues and carbohydrates linked to them. These results demonstrate that LH3 is indispensable for biosynthesis of type IV collagen and for BM stability during early development and that loss of LH3's functions leads to embryonic lethality. We propose that the premature aggregation of collagen IV is due to the absence of the hydroxylysine-linked carbohydrates, which thus play an essential role in its supramolecular assembly. L ysyl hydroxylase (LH, EC 1.14.11.4, Plod) catalyzes the hydroxylation of lysine residues in -X-Lys-Gly-triplets in collagens and other proteins with collagen-like sequences (1). The hydroxylysine residues formed have two important functions: (i) they serve as attachment sites for carbohydrates, either monosaccharide galactose or disaccharide glucosylgalactose, and (ii) they have an important role in the stabilization of intermolecular collagen crosslinks that provide tensile strength and mechanical stability for the collagen fibrils. The extent of lysine hydroxylation and hydroxylysine glycosylation varies between collagen types. Nearly 90% of all lysines are hydroxylated and hydroxylysines glycosylated in types IV and VI collagen, whereas Ͻ20% of the lysines are hydroxylated in type III collagen (2). The functions of the hydroxylysine-linked carbohydrates are poorly understood, but in the case of fibril-forming collagens, they are thought to regulate the formation and morphology of the fibrils (1, 3).Three human LH isoenzymes have been identified (1, 4-6). Mutations in the LH1 gene lead to the kyphoscoliotic type of Ehlers-Danlos syndrome, which is characterized by scoliosis, joint laxity, skin fragility, ocular manifestations, and severe muscle hypotonia (7-9). Mutations in the LH2 gene have recently been reported in two families with Bruck syndrome, which is characterized by fragile bones, joint contractures, scoliosis, and osteoporosis (10).No mutations have been characterized in the gene for the isoenzyme LH3, and its in vivo functions have not yet been elucidated. This protein differs from the other two lysyl hydroxylase isoenzymes in that it also possesses relatively low levels...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Rautavuoma

Takaluoma

Sormunen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

102

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“…Very surprisingly, however, recombinant LH3 was also found to possess collagen glucosyltransferase activity but not LH1 or LH2 (17). No data are available on the level of this activity in the LH3 polypeptide nor on the possible biological significance of the finding (17).…”

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confidence: 97%

Characterization of Three Fragments That Constitute the Monomers of the Human Lysyl Hydroxylase Isoenzymes 1–3

Rautavuoma

Takaluoma

Passoja

et al. 2002

Journal of Biological Chemistry

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Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80 -85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä , R. (2000) J. Biol. Chem. 275, 36158 -36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.

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“…9 LH3 differs from LH1 and LH2 in that it is multifunctional and able to catalyze, in addition to Lys hydroxylation, sugar transfer reactions, the subsequent steps in the formation of glucosylgalactosyl-Hyl residues. [12][13][14] Only one isoform for LH is present in lower species such as Caenorhabditis elegans. 9,15 This ancestral C. elegans LH is also able to glycosylate Hyl residues and, thus, is functionally similar to LH3.…”

Section: Introductionmentioning

confidence: 99%

Development of a convenient peptide‐based assay for lysyl hydroxylase

Čudić¹,

Patel²,

Lauer‐Fields³

et al. 2008

Biopolymers

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Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C‐5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2‐oxoglutarate; the latter is decarboxylated to form succinate and CO2. To develop a convenient, RP‐HPLC based LH assay, we used Fmoc solid‐phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP‐HPLC, MALDI‐TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP‐HPLC conditions. The best results were achieved using peptide derivatization (1‐anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP‐HPLC analysis. The products (di‐ and tetra‐substituted Lys‐ and Hyl‐containing peptides) were well resolved by RP‐HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP‐HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi‐step, require radio‐labeled substrates, and/or measure depletion of 2‐oxoglutarate or formation of CO2. Since the LH reaction with 2‐oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 330–338, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Lysyl Hydroxylase 3 Is a Multifunctional Protein Possessing Collagen Glucosyltransferase Activity

Cited by 126 publications

References 18 publications

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Characterization of Three Fragments That Constitute the Monomers of the Human Lysyl Hydroxylase Isoenzymes 1–3

Development of a convenient peptide‐based assay for lysyl hydroxylase

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