Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Salo, Antti M.; Wang, Chunguang; Sipilä, Laura; Sormunen, Raija; Vapola, Miia; Kervinen, P.; Ruotsalainen, Heli; Heikkinen, Jari; Myllylä, Raili

doi:10.1002/jcp.20596

Cited by 67 publications

(90 citation statements)

References 60 publications

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“…Localization can be tissue-specific, may require proteolysis, and may be regulated by mechanisms such as alternative splicing or putative RNA editing (24,26,27). In this study, native skeletal muscle FKRP was detected as a smaller ϳ50-kDa protein in skeletal muscle (predicted core protein ϳ55 kDa).…”

Section: Discussionmentioning

confidence: 55%

“…FKRP as a molecular chaperone for DG processing and/or targeting, or vice versa, is an intriguing possibility. Whether FKRP activity is required at the sarcolemma for normal muscle function is unclear; however, known glycosyltransferases may possess enzymatic or lectin binding activity at the cell surface (23)(24)(25)30). Overall, our data indicate that FKRP is present at the muscle cell surface, associates with the DGC, and may have a unique role in the ␣DG processing pathway.…”

Section: Discussionmentioning

confidence: 85%

“…Cell surface and extracellular localization has been described for several glycosyltransferases and related enzymes (23)(24)(25)(26)(27). Localization can be tissue-specific, may require proteolysis, and may be regulated by mechanisms such as alternative splicing or putative RNA editing (24,26,27).…”

Section: Discussionmentioning

confidence: 99%

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Fukutin-related Protein Associates with the Sarcolemmal Dystrophin-Glycoprotein Complex

Beedle

Nienaber

Campbell

2007

Journal of Biological Chemistry

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Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex. Mutations in FKRP3 lead to the allelic muscular dystrophies, congenital muscular dystrophy 1C and limb girdle muscular dystrophy 2I, characterized by progressive muscle weakness with variable heart, respiratory, and brain involvement (1-3). Although the specific function of FKRP is unclear, FKRP and its closest known homolog fukutin share sequence homology with phosphoryl ligand transferases and contain DXD domains common to some glycosyltransferases (4, 5). In addition, FKRPassociated muscular dystrophies fall into a growing family of "dystroglycanopathies," which exhibit reduced glycosylation of membrane-associated ␣DG (6). Extracellular ␣DG and the transmembrane-spanning ␤DG bind to dystrophin, sarcoglycans, and other proteins to form the dystrophin-glycoprotein complex (DGC), which serves as a critical structural link between the cell cytoskeleton, the sarcolemma, and the extracellular basement membrane. ␣DG glycans, detected by antibody IIH6, mediate the interaction between the DGC and extracellular matrix proteins that contain laminin LG domains. Therefore, reduced ␣DG glycosylation may weaken the cell to matrix link, increasing structural instability and disease (6).Previous studies have examined the localization of FKRP in a spectrum of cultured cells (e.g. COS7, SH-SY5Y, C2C12). The variable results suggested that FKRP is a resident of the Golgi, the rough endoplasmic reticulum, or perinuclear regions (7-11). In this study, we have examined FKRP protein complexes and their location in skeletal muscle of wild-type and dystrophic mice. We report that FKRP is localized at the muscle sarcolemma and that it co-fractionates with the DGC. Furthermore, disruption of the DGC (by alkaline treatment or genetic deletion) revealed that FKRP sedimentation and localization are dependent on...

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 85%

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Fukutin-related Protein Associates with the Sarcolemmal Dystrophin-Glycoprotein Complex

Beedle

Nienaber

Campbell

2007

Journal of Biological Chemistry

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“…2.4.1.66) activities in vitro (11)(12)(13). LH3 is functional in both the endoplasmic reticulum and extracellular space (14), and loss of LH3 leads to early embryonic lethality (15,16). We have reported recently that LH3 is also multifunctional in vivo and that especially the glucosyltransferase activity of LH3 is essential for normal embryonic development (16).…”

mentioning

confidence: 99%

Secretion and Assembly of Type IV and VI Collagens Depend on Glycosylation of Hydroxylysines

Sipilä

Ruotsalainen

Sormunen

et al. 2007

Journal of Biological Chemistry

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Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. Collagen biosynthesis contains many co-and post-translational modifications that are unique to collagenous proteins. These modifications include hydroxylation of lysyl residues and their further glycosylation to galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues. Lysyl hydroxylase (LH 2 ; E.C. 1.14.11.4) catalyzes the hydroxylation of lysyl residues in the Y-position of the repeating collagen Gly-X-Y triplets (1, 2), and three lysyl hydroxylase isoforms have been characterized: LH1, LH2, and LH3 (3-10).LH3 is a multifunctional enzyme containing, in addition to lysyl hydroxylase activity, collagen galactosyltransferase (E.C. 2.4.1.50) and glucosyltransferase (E.C. 2.4.1.66) activities in vitro (11-13). LH3 is functional in both the endoplasmic reticulum and extracellular space (14), and loss of LH3 leads to early embryonic lethality (15, 16). We have reported recently that LH3 is also multifunctional in vivo and that especially the glucosyltransferase activity of LH3 is essential for normal embryonic development (16).The amount of lysine hydroxylation and glycosylation varies between different collagen types. Type IV and VI collagens are highly hydroxylated and glycosylated, whereas fibrillar collagens, especially types I and III, have much lower levels of these modifications (1, 17, 18). The hydroxylysyl residues have an important role in collagen cross-linking (19,20), but the function of the glycosylation of the hydroxylysyl residues is still poorly understood. The role of glycosylated hydroxylysines in the formation and morphology of collagen fibrils has been debated (21, 22), and we have shown that they are necessary for the correct basement membrane localization of type IV collagen (16).We produced previously three different mouse lines with a manipulated LH3 gene (16). The mice with no or a very low level of the LH3 protein, thus lacking the lysyl hydroxylase and glucosyltransferase activities of LH3, had an embryonic lethal phenotype. The mouse line (LH mutant) with a point mutation that specifically destroys the LH activity of LH3 without affecting glycosyltransferase activities (11) was viable, but showed ultrastructural alterations (16). In this study, we used LH3 knock-out and LH mutant mice and fibroblasts derived from them as a model to investigate the functions of glycosylated hydroxylysines of collagens. This work provides new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion of type IV collagen and in the tetramerization and *

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“…9,15 This ancestral C. elegans LH is also able to glycosylate Hyl residues and, thus, is functionally similar to LH3. 9,13 Recently, Salo et al 16 found that LH3 is located in two compartments in tissues, in the endoplasmic reticulum and the extracellular space, and the partitioning varies with tissue type. Thus, LH3 may be functional both inside and outside of the cell.…”

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 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Cited by 67 publications

References 60 publications

Fukutin-related Protein Associates with the Sarcolemmal Dystrophin-Glycoprotein Complex

Fukutin-related Protein Associates with the Sarcolemmal Dystrophin-Glycoprotein Complex

Secretion and Assembly of Type IV and VI Collagens Depend on Glycosylation of Hydroxylysines

Development of a convenient peptide‐based assay for lysyl hydroxylase

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