Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix

• DOI: 10.1074/jbc.m109.038190
• Publication Date: 2009-08-21T00:14:00Z
• Authors: Maija Risteli; Heli Ruotsalainen; Antti M. Salo; Raija Sormunen; Laura Sipilä; Naomi L. Baker; Shireen R. Lamandé; Leena Vimpari-Kau...; Raili Myllylä
• Keywords: Epidermolysis bullosa simplex; Collagen VI

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of extracellular matrix (ECM) cytoskeleton. Deposition ECM was affected heterozygous knock-out mouse embryonic fibroblasts (MEF+/−) skin collected from member Finnish epidermolysis bullosa simplex (EBS) family known to be deficient GGT activity. show deficiency due transcriptional defect one allele. The abnormalities also lead defects arrangement cytoskeleton both cell lines. Ultrastructural were observed mice indicating that even moderate decrease has deleterious consequences vivo. null allele EBS resulting matrix, similar those found MEF+/−, may explain correlation between severity phenotype activity reported this family. (LH) 2The abbreviations used are: LHlysyl hydroxylaseGGTglucosyltransferaseMEFmouse fibroblastsEBSepidermolysis simplexECMextracellular matrix. catalyzes post-translational formation hydroxylysines -X-Lys-Gly- sequences collagens other proteins with collagen-like domains (1Kivirikko K.I. Myllylä R. Pihlajaniemi T. Post-translational Modifications Proteins.in: Harding J.J. Crabbe J.C. CRC Press, Boca Raton, FL1992: 1-51Google Scholar, 2Kivirikko Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). Three isoforms (LH1, LH2, LH3) have been identified human, mouse, rat, zebrafish (3Hautala Byers M.G. Eddy R.L. Shows T.B. Kivirikko Genomics. 1992; 13: 62-69Crossref PubMed Scopus (115) 4Valtavaara M. Papponen H. Pirttilä A.M. Hiltunen K. Helander J. Chem. 1997; 272: 6831-6834Abstract Full Text PDF (109) 5Valtavaara Szpirer C. 273: 12881-12886Abstract (106) 6Passoja Rautavuoma Ala-Kokko L. Kosonen Proc. Natl. Acad. Sci. U.S.A. 95: 10482-10486Crossref (91) 7Ruotsalainen Sipilä Kerkelä E. Pospiech Matrix 1999; 18: 325-329Crossref (43) 8Mercer D.K. Nicol P.F. Kimbembe Robins S.P. Biochem. Biophys. Res. Commun. 2003; 307: 803-809Crossref (86) 9Schneider V.A. Granato Neuron. 2006; 50: 683-695Abstract (73) 10Schneider 2007; 26: 12-19Crossref (12) In addition, LH2 two alternatively spliced forms, LH2a (short) LH2b (long) (11Yeowell H.N. Walker L.C. 179-187Crossref (74) Several mutations LH1 gene (PLOD1) patients Ehlers-Danlos syndrome (EDS) type VIA, heritable disorder characterized by kyphoscoliosis, joint laxity, fragility, muscle hypotonia (12Yeowell Genet. Metab. 2000; 71: 212-224Crossref (127) 13Steinmann B. Eyre D.R. Superti-Furga A. Connective Tissue Its Heritable Disorders.in: Royce P.M. Steinmann 2nd Ed. Wiley-Liss Inc., New York2002: 431-523Crossref EDS VIA data indicate hydroxylates helical cross-linking lysines I bone II cartilage (14Eyre D. Shao P. Weis M.A. 2002; 76: 211-216Crossref (49) 15Uzawa Yeowell Yamamoto Mochida Y. Tanzawa Yamauchi 305: 484-487Crossref (21) Patients Bruck syndrome, contractures, fragile bones, osteoporosis, (PLOD2) result complete absence telopeptide hydroxylysine residues (16van der Slot A.J. Zuurmond Bardoel A.F. Wijmenga Pruijs H.E. Sillence D.O. Brinckmann Abraham D.J. Black C.M. Verzijl N. DeGroot Hanemaaijer TeKoppele J.M. Huizinga T.W. Bank R.A. 278: 40967-40972Abstract (299) 17Ha-Vinh Alanay Campos-Xavier A.B. Zankl Bonafé Am. Med. 2004; 131: 115-120Crossref (151) overexpression linked increase content telopeptides seen fibrotic disorders (18van van den Bogaerdt Ulrich M.M. Middelkoop Boers W. Karel Ronday 23: 251-257Crossref (156) differs it possesses, addition (6Passoja 19Valtavaara (92) Scholar), hydroxylysyl galactosyltransferase galactosylhydroxylysyl activities (20Heikkinen Risteli Wang Latvala Rossi Valtavaara 275: 36158-36163Abstract (108) 21Wang Luosujärvi Heikkinen Uitto 21: 559-566Crossref (90) 22Myllylä Juffer Lampela O. Ruotsalainen Salo Cell. Physiol. 212: 323-329Crossref (89) thus able catalyze glucosylgalactosylhydroxylysine residues. Recent studies located not only endoplasmic reticulum but space, serum originates (23Salo Sormunen Vapola Kervinen 207: 644-653Crossref (56) Furthermore, demonstrate loss leads lethality disruption basement membranes (24Rautavuoma Takaluoma Myllyharju Soininen 101: 14120-14125Crossref (93) 25Ruotsalainen Mercer Aszodi Fässler Cell 119: 625-635Crossref Analyses embryos glycosylation prevents assembly secretion IV VI (26Sipilä Baker N.L. Lamandé S.R. Sado 282: 33381-33388Abstract (61) Recently, human (PLOD3) shown cause severe connective tissue features overlap number (27Salo Cox Farndon Moss Grindulis Hum. 2008; 83: 495-503Abstract (83) presence intra- space drastic membrane when absent during early development suggest plays deposition ECM. To examine more detail, we investigated affects partial on cells tissues. examined at ultrastructural level as well model deficiency, exhibit dominant EBS, accompanied markedly decreased urinary excretion glucosylgalactosylhydroxylysine, earlier (28Savolainen E.R. Kero Engl. 1981; 304: 197-204Crossref (37) Among families tested, activity, feature unique does represent typical finding Savolainen co-workers Scholar) correlated disease. re-examined patient determined Our results amount intracellular substantial causes changes cytoskeletal cell. This tissues mice, cells, representing conditions where produces LH3. reveal A fibroblast culture established biopsy specimen taken belonging detailed elsewhere marked 10 pedigree study. One control kindly provided Dr. Kaisa Tasanen-Määttä others locally established. All samples obtained after informed consent, according Declaration Helsinki. Heterozygous crosses derive described maintained 5% CO2 Dulbecco's modified Eagle's medium Glutamax (Invitrogen) supplemented 10% fetal calf (Promocell), penicillin/streptomycin, 50 μg/ml ascorbic acid. production previously (25Ruotsalainen animal experiments approved Animal Care Use Committee University Oulu. For transmission electron microscopy, newborn adult fixed 1% glutaraldehyde, 4% formaldehyde 0.1 m phosphate buffer (pH 7.4), post-fixed osmium tetroxide, dehydrated acetone, embedded Epon LX 112 (Ladd Research Industries, Williston, VT). Thin sections cut Leica Ultracut UCT ultramicrotome Philips CM100 microscope. diameters 200–250 fibrils measured thin three wild-type using ITEM Soft Imaging Solutions (Olympus). Statistical analysis performed Student's t test. immunoelectron confluent 58-cm2 dishes paraformaldehyde 7.3), into gelatin, immersed 2.3 sucrose. cryosections incubated polyclonal antibody (PLOD3 antibody, ProteinTech Group, Inc.) then protein A-gold complex. above. Cells dish homogenized (4Valtavaara Culture 72 h concentrated about 250 μl Amicon Ultra 10k centrifugal filter device (Millipore). assay based transfer tritium-labeled sugar UDP-glucose (139 Ci/mol) galactosyl gelatin substrate (29Kivirikko Methods 1982; 82: 245-304Crossref (324) 30Myllylä Eur. 1975; 52: 401-410Crossref Total RNA isolated cultured TRIzol plus purification kit (Invitrogen). cDNA synthesized total cloned avian myeloblastosis virus first-strand synthesis random hexamer or oligo(dT)20-primed reactions. expression detected TaqMan (Hs00153670_m1, PLOD3) (Applied Biosystems) Universal PCR Master Mix 7500 real time system relative standard curve method. eukaryotic 18 S rRNA endogenous normalize quantities expression. Genomic DNA proteinase K phenol extraction (31Sambrook Russel D.W. Molecular Cloning, Laboratory Manual. 3rd Cold Spring Harbor Harbor, NY2001Google amplification promoter sequence exon 1-specific oligonucleotides covering 1043 nucleotides upstream translation start site (32Rautavuoma Passoja Helaakoski 19: 73-79Crossref (9) (AF207069), amplified LH3-specific entire coding region 303 5′- 259 3′-untranslated regions (5Valtavaara (NM_001084). overlapping fragments directly sequenced DYEnamic ET terminator cycle sequencing premix (GE Healthcare) ABI Prism 377 sequencer (PerkinElmer Life Sciences). grown glass coverslips phosphate-buffered saline. 95% Ethanol, acetic acid tubulin vimentin analyses. After blocking bovine albumin, 0.1% saponin saline, monoclonal anti-vimentin (Affinity BioReagents), (Sigma), anti-α-tubulin (Sigma) primary antibodies diluted solution. fibronectin experiments, rabbit anti-collagen (Rockland), anti-human saponin, nuclei stained Hoechst 33258 (Sigma). Alexa Fluor 488-conjugated anti-rabbit IgG, anti-mouse anti-goat IgG secondary detection. actin microfilament staining, permeabilized Triton X-100 568 phalloidin Immu-Mount (Thermo Shandon) Olympus epifluorescence microscope Fluoview 1000 confocal partially purified UDP-hexanolamine-agarose Equal amounts soluble equal volumes 72-h media analyzed immunoblots Inc.). analysis, washed twice scraped homogenization (0.2 NaCl, glycine, X-100, μm dithiothreitol, mm EDTA, 20 Tris, pH 7.5) lysed brief sonication. debris removed centrifugation 15,000 × g 15 min. serum-free trichloroacetic acid-precipitated redissolved SDS sample buffer. samples, lysate solubilized precipitate, separated 7.5% SDS-PAGE transferred Immobilon-P polyvinylidene difluoride Nonspecific binding blocked nonfat milk powder Tris-buffered saline-Tween. (Rockland) further horseradish peroxidase-conjugated (P.A.R.I.S) antibody. Immunocomplexes visualized ECL+ reagent Kodak X-AR films. Types α chain levels quantified ImageQuant 5.2 software distribution compared medium. Type biosynthetically labeled [35S]methionine, immunoprecipitated, (33Lamandé Mörgelin Adams N.E. Selan Allen 281: 16607-16614Abstract (52) LH mutant showed alterations their 26Sipilä Therefore these microscopy. had no obvious (data shown). However, dermis appeared looser edematous than dermis. Analysis cross-sections mean fibril diameter significantly (p < 0.001) (Fig. 1, B) wild-type, being 52 ± 61 8 nm, respectively. frequency variable differed 1B). skin, variation genotypes. contained small higher range wider although statistically significant 1C). These fibrillogenesis reduced glucosylgalactosylhydroxylysine. determine whether could observations. 2A) are consistent previous showing (about 70% control) 10. 42% controls. (80% normal (MEF+/+), Fig. 2B) our (MEF+/−). MEF+/− MEF+/+, reduction GGT/LH3 inside cells. fibroblasts. main molecule responsible correlates assayed immunoblotting On immunoblots, 3A). molecular weight did differ suggesting processing patient. microscopy 3B), line confirming When gold particles 4.4-μm2 area counted 30 fewer particles, 57% controls, fibroblasts, revealing Taken together, there differences produced quantity LH3, either protein, pronounced study reason quantitative PCR, 1 sequenced. mRNA 67% (S.D. 11) level. Sequencing any genomic 3C), A195G A434G upstream, C882T A1011G downstream transcription PLOD3 (AF207096) Changes positions A195G, A434G, healthy which reveals them polymorphic changes. change present A376 (NM_001084) 3C). T position 247 corresponding alleles transcribed agreement analysis. findings low (PLOD3). recent indicated glycosylated tetramerization impaired underglycosylation aggregation As earlier, controls (34Sabatelli Bonaldo Lattanzi G. Braghetta Bergamin Capanni Mattioli Columbaro Ognibene Pepe Bertini Merlini Maraldi N.M. Squarzoni S. 2001; 20: 475-486Crossref (97) furthermore, play (35Li Van Den Diepstraten D'Souza S.J. Chan B.M. Pickering J.G. Pathol. 163: 1045-1056Abstract 36Minamitani Ikuta Saito Takebe Sato Sawa Nishimura Nakamura F. Takahashi Ariga Matsumoto Exp. 298: 305-315Crossref (102) cultures. Immunofluorescence staining carried out non-permeabilized days confluence observe components, collagen, fibronectin, abnormal (MEF+/+) 4). formed fine slightly interconnected three-dimensional network 4B), whereas thick heavily cross-linked MEF+/+ 4A). Subsequently, 4F). arranged nest-like pattern 4E), whereas, few interconnections 4J); short faint, differing 4I). 4, D, H, L) revealed arrangements collagens.