Identification of PLOD2 as Telopeptide Lysyl Hydroxylase, an Important Enzyme in Fibrosis

Abstract

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes. The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes. The biosynthesis of collagen molecules involves several intracellular post-translational modifications followed by excretion and extracellular aggregation of the collagen molecules into fibrils, which are subsequently stabilized by intermolecular cross-links (1von der Mark K. Seibel M.J. Robins S.P. Bilezikian J.P. Dynamics of Bone and Cartilage. Academic Press, London1999: 3-30Google Scholar, 2Robins S.P. Seibel M.J. Robins S.P. Bilezikian J.P. Dynamics of Bone and Cartilage. Academic Press, London1999: 31-42Google Scholar). Two related routes are responsible for the formation of these collagen cross-links, namely the allysine route, in which a lysine residue in the telopeptide is converted by lysyl oxidase into the aldehyde allysine, and the hydroxyallysine route, in which a hydroxylysine residue in the telopeptide is converted into the aldehyde hydroxyallysine. Subsequently, the allysine or the hydroxyallysine reacts with a Lys or Hyl residue in the triple helix to form di-, tri-, or tetrafunctional cross-links (3Eyre D.R. Paz M.A. Gallop P.M. Annu. Rev. Biochem. 1984; 53: 717-748Crossref PubMed Google Scholar, 4Herbage D. Le Lous M. Cohen-Solal L. Bazin S. Front. Matrix Biol. 1985; 10: 59-91Google Scholar, 5Reiser K. McCormick R.J. Rucker R.B. FASEB J. 1992; 6: 2439-2449Crossref PubMed Scopus (358) Google Scholar, 6Robins S.P. Methods Biochem. Anal. 1982; 28: 329-379Crossref PubMed Google Scholar). The mature cross-links hydroxylysylpyridinoline or lysylpyridinoline are formed via the hydroxyallysine route and occur in a variety of connective tissues such as bone, tendon, ligaments, and cartilage (7Eyre D.R. Koob T.J. Van Ness K.P. Anal. Biochem. 1984; 137: 380-388Crossref PubMed Scopus (637) Google Scholar). In contrast, collagen in the skin is mainly cross-linked via the allysine route. Interestingly, in fibrotic skin (lipodermatosclerosis, keloid) and organ fibrosis (lung, liver), which is characterized by an excessive accumulation of collagen, an increase in cross-links derived from the hydroxyallysine route is found (8Brinckmann J. Notbohm H. Tronnier M. Acil Y. Fietzek P.P. Schmeller W. Muller P.K. Batge B. J. Invest. Dermatol. 1999; 113: 617-621Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Brinckmann J. Neess C.M. Gaber Y. Sobhi H. Notbohm H. Hunzelmann N. Fietzek P.P. Muller P.K. Risteli J. Gebker R. Scharffetter-Kochanek K. J. Investig. Dermatol. 2001; 117: 269-273Abstract Full Text Full Text PDF PubMed Google Scholar, 10Last J.A. King T.E. Nerlich A.G. Reiser K.M. Am. Rev. Respir. Dis. 1990; 141: 307-313Crossref PubMed Scopus (49) Google Scholar, 11Ricard-Blum S. Bresson-Hadni S. Vuitton D.A. Ville G. Grimaud J.A. Hepatology. 1992; 15: 599-602Crossref PubMed Scopus (78) Google Scholar, 12Ricard-Blum S. Esterre P. Grimaud J.A. Cell. Mol. Biol. 1993; 39: 723-727PubMed Google Scholar, 13Uzawa K. Marshall M.K. Katz E.P. Tanzawa H. Yeowell H.N. Yamauchi M. Biochem. Biophys. Res. Commun. 1998; 249: 652-655Crossref PubMed Scopus (34) Google Scholar, 14Bailey A.J. Bazin S. Sims T.J. Le Lous M. Nicoletis C. Delaunay A. Biochim. Biophys. Acta. 1975; 405: 412-421Crossref PubMed Scopus (228) Google Scholar, 15Bailey A.J. Light N.D. CIBA Found. Symp. 1985; 114: 80-96PubMed Google Scholar). It has been shown that the amount of hydroxyallysine-derived cross-links is related to the irreversible accumulation of collagen in fibrotic tissues, indicating that collagen containing hydroxyallysine-derived cross-links is more difficult to degrade than collagen containing allysine-derived cross-links (10Last J.A. King T.E. Nerlich A.G. Reiser K.M. Am. Rev. Respir. Dis. 1990; 141: 307-313Crossref PubMed Scopus (49) Google Scholar, 11Ricard-Blum S. Bresson-Hadni S. Vuitton D.A. Ville G. Grimaud J.A. Hepatology. 1992; 15: 599-602Crossref PubMed Scopus (78) Google Scholar, 12Ricard-Blum S. Esterre P. Grimaud J.A. Cell. Mol. Biol. 1993; 39: 723-727PubMed Google Scholar, 14Bailey A.J. Bazin S. Sims T.J. Le Lous M. Nicoletis C. Delaunay A. Biochim. Biophys. Acta. 1975; 405: 412-421Crossref PubMed Scopus (228) Google Scholar, 15Bailey A.J. Light N.D. CIBA Found. Symp. 1985; 114: 80-96PubMed Google Scholar). Inhibition of the formation of hydroxyallysine-derived cross-links in fibrosis is therefore likely to result in the formation of collagen that is easier to degrade, thereby preventing the unwanted collagen accumulation. The increase in hydroxyallysine-derived cross-links in fibrosis is the result of an overhydroxylation of lysine residues within the collagen telopeptides. The enzyme catalyzing the conversion of Lys into Hyl is lysyl hydroxylase (EC 1.14.11.4) (16Hautala T. Byers M.G. Eddy R.L. Shows T.B. Kivirikko K.I. Myllyla R. Genomics. 1992; 13: 62-69Crossref PubMed Scopus (112) Google Scholar, 17Heikkinen J. Toppinen T. Yeowell H. Krieg T. Steinmann B. Kivirikko K.I. Myllyla R. Am. J. Hum. Genet. 1997; 60: 48-56PubMed Google Scholar, 18Yeowell H.N. Walker L.C. Proc. Assoc. Am. Physicians. 1997; 109: 383-396PubMed Google Scholar). The lysyl hydroxylase family consists of lysyl hydroxylase 1 (LH1), 1The abbreviations used are: LH, lysyl hydroxylase; BS, Bruck syndrome; COL1A2, collagen type 1 α2 chain; LOX, lysyl oxidase; PLOD, procollagen-lysine, 2-oxoglutarate 5-dioxygenase; SSc, systemic sclerosis; TLH, telopeptide lysyl hydroxylase.1The abbreviations used are: LH, lysyl hydroxylase; BS, Bruck syndrome; COL1A2, collagen type 1 α2 chain; LOX, lysyl oxidase; PLOD, procollagen-lysine, 2-oxoglutarate 5-dioxygenase; SSc, systemic sclerosis; TLH, telopeptide lysyl hydroxylase. LH2, and LH3 encoded by procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), PLOD2, and PLOD3, respectively (16Hautala T. Byers M.G. Eddy R.L. Shows T.B. Kivirikko K.I. Myllyla R. Genomics. 1992; 13: 62-69Crossref PubMed Scopus (112) Google Scholar, 19Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (89) Google Scholar, 20Valtavaara M. Papponen H. Pirttila A.M. Hiltunen K. Helander H. Myllyla R. J. Biol. Chem. 1997; 272: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Valtavaara M. Szpirer C. Szpirer J. Myllyla R. J. Biol. Chem. 1998; 273: 12881-12886Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Alternative RNA splicing has been described for PLOD2 only, resulting in the splice variants LH2a and LH2b (LH2b contains an extra exon; Ref. 22Yeowell H.N. Walker L.C. Matrix Biol. 1999; 18: 179-187Crossref PubMed Scopus (70) Google Scholar). The Ehlers-Danlos syndrome type VI is characterized by mutations in PLOD1. Collagen of these patients shows an absence of hydroxylated lysine residues within the triple helix in combination with normal levels of hydroxylated lysine residues within the telopeptides. Therefore, it can be concluded that LH1 catalyzes the conversion of triple helical lysine residues into hydroxylysines (23Yeowell H.N. Walker L.C. Mol. Genet. Metab. 2000; 71: 212-224Crossref PubMed Scopus (127) Google Scholar, 24Steinmann B. Eyre D.R. Shao P. Am. J. Hum. Genet. 1995; 57: 1505-1508PubMed Google Scholar). The substrate specificity of LH2 and LH3 has not yet been elucidated, and to date no disease has been associated with defects in LH2 or LH3. Bruck syndrome (BS; Online Mendelian Inheritance in Man (OMIM) accession number 259450), an autosomal recessive disease, is characterized by osteoporosis, joint contractures at birth, fragile bones, and short stature (25Brenner R.E. Vetter U. Stoss H. Muller P.K. Teller W.M. Eur. J. Pediatr. 1993; 152: 505-508Crossref PubMed Scopus (39) Google Scholar, 26Breslau-Siderius E.J. Engelbert R.H. Pals G. van der Sluijs J.A. J. Pediatr. Orthop. B. 1998; 7: 35-38Crossref PubMed Scopus (45) Google Scholar, 27Leroy J.G. Nuytinck L. De Paepe A. De Rammelaere M. Gillerot Y. Verloes A. Loeys B. De Groote W. Pediatr. Radiol. 1998; 28: 781-789Crossref PubMed Scopus (19) Google Scholar, 28McPherson E. Clemens M. Am. J. Med. Genet. 1997; 70: 28-31Crossref PubMed Google Scholar). Biochemical analysis of the bone of BS patients revealed an underhydroxylation of lysine residues in the telopeptides of collagen type I, whereas hydroxylation of lysine residues in the triple helix is normal (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar). Together, the Ehlers-Danlos syndrome type VI and the Bruck syndrome imply that a lysyl hydroxylase must exist that specifically hydroxylates the telopeptides and that the activity of such a telopeptide lysyl hydroxylase (TLH) is decreased in BS bone. The identity of TLH had not yet been discovered. Here we show that the gene defect in two BS families is a mutation in PLOD2, showing that PLOD2 encodes TLH. The importance of TLH in fibrotic processes is demonstrated by the highly increased expression of TLH in fibroblasts cultured from the fibrotic skin of systemic sclerosis (SSc) patients. Furthermore, higher levels of pyridinoline cross-links were found in the extracellular matrix deposited by the SSc fibroblasts, which shows the link between increased TLH expression and the shift to the hydroxyallysine cross-linking pathway. Patients—In this study, three families with children diagnosed as Bruck syndrome patients were investigated. Two unrelated Kurdish families (families FH and PM), both consanguineous, were described earlier (cases 1, 2, and 3, and 7 and 8, respectively) (26Breslau-Siderius E.J. Engelbert R.H. Pals G. van der Sluijs J.A. J. Pediatr. Orthop. B. 1998; 7: 35-38Crossref PubMed Scopus (45) Google Scholar); the third family (family DR) has not yet been described. This Australian family, where the parents are not consanguineous, had two affected boys deceased prenatally, one affected girl deceased at 2 years, and one surviving unaffected 9-year-old girl. All these BS patients show decreased amounts of pyridinoline cross-links in bone. 2A. J. van der Slot, A.-M. Zuurmond, H. E. H. Pruijs, D. O. Sillence, J. DeGroot, T. W. J. Huizinga, and R. A. Bank, submitted for publication. Genotyping—Blood samples of the three families were collected, and genomic DNA was prepared by standard methods. Microsatellite markers, obtained from the Marshfield screening set version 6, were amplified as described earlier (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar). Genotypes of the previously localized BS locus, which is 17p12 (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar), and the chromosomal region of PLOD2, which is 3q23-q24 (30Szpirer C. Szpirer J. Riviere M. Vanvooren P. Valtavaara M. Myllyla R. Mamm. Genome. 1997; 8: 707-708Crossref PubMed Scopus (22) Google Scholar), were assigned. Mutation Analysis—The exon/intron organization of PLOD2 has not been described. PLOD1 and PLOD3, encoding two other members of the lysyl hydroxylase family, both consist of 19 exons (31Heikkinen J. Hautala T. Kivirikko K.I. Myllyla R. Genomics. 1994; 24: 464-471Crossref PubMed Scopus (46) Google Scholar, 32Rautavuoma K. Passoja K. Helaakoski T. Kivirikko K.I. Matrix Biol. 2000; 19: 73-79Crossref PubMed Scopus (9) Google Scholar). The exon/intron boundaries of both genes are identical. Analysis of clones RP11-758I14 and exon/intron boundaries for In PLOD2 shows an extra exon H.N. Walker L.C. Matrix Biol. 1999; 18: 179-187Crossref PubMed Scopus (70) Google Scholar), exon which is not found in PLOD1 or DNA in exons and exon/intron boundaries of PLOD2, were to the the exon/intron in a of the region was amplified was genomic DNA from Bruck syndrome unaffected and a of a at of for 1 a as in for or and for an of at was as for the and were the for mutation for exon is for exon 19 is for exon is for exon 19 is in a from skin of patients with SSc were by methods. were obtained from from were in with and were of and used for RNA RNA was the Subsequently, the RNA was into and to of was for the α2 of collagen type 1 and lysyl oxidase was for the to for in the amount of was amplified and and respectively) in a of containing of and 1 of was in an and of a at followed by of for for and for were version for in a for in a of LH2a and of and SSc fibroblasts was to LH2a and LH2b LH2a and LH2b were and in a of containing and 1 of was in a and of a at followed by of for for and for of of amplified were to in a containing were extracellular matrix obtained from fibroblasts cultured for in the of cross-links were in of samples by R.A. B. N. De J.A. Tekoppele J.M. J. Sci. 1997; PubMed Scopus Google Scholar, S.P. A. N. Chem. PubMed Scopus (89) Google Scholar). analysis was analysis and data are as standard and were by are as PLOD2, PLOD3, COL1A2, and LOX, Analysis of BS the gene encoding TLH, the gene defect in BS patients was In a we the BS an region 17p12 (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar). that a family was for of the of the the two other diagnosed BS families (family and family were for to analysis the of both families to This in to a BS a BS chromosomal region that the region for the BS is as PLOD2 encoding LH2 is in that region (30Szpirer C. Szpirer J. Riviere M. Vanvooren P. Valtavaara M. Myllyla R. Mamm. Genome. 1997; 8: 707-708Crossref PubMed Scopus (22) Google Scholar). This was the of a increase of LH2 expression and hydroxyallysine-derived cross-links in the of in K. T. D.A. Yamauchi M. J. Bone Res. 1999; PubMed Scopus Google Scholar), which that LH2 catalyzes the conversion of the telopeptide lysine residue into analysis of family and family revealed that both families the of 1, a and Mutation Analysis of BS that the BS in the patients in family and family is to mutations in PLOD2, a mutation analysis was In both patients of family a missense mutation in exon 17 of was resulting in a at and and parents and the were of the mutation In the exon a missense mutation of was in both patients of family resulting in a at and and The parents were for the mutation mutation was in a of of thereby the that these mutations Mutation analysis of patients from the BS family (family showing to 17p12 (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google not mutations in the exon/intron and the region of TLH in SSc BS that the substrate of LH2 is the lysine residue within the collagen we were in the expression of LH2 is increased in in fibrotic as the collagen deposited in fibrotic shows an increased hydroxylation of the telopeptides. analysis of mRNA from SSc skin fibroblasts revealed that the expression of LH2b mRNA was highly increased in these fibroblasts with SSc patients Interestingly, LH1 SSc patients and LH3 SSc patients expression levels were of LH2a mRNA was both in and SSc patients In with data J. Invest. PubMed Scopus Google Scholar, L. A. Myllyla R. Krieg T. A. J. Invest. Dermatol. 1985; Full Text PDF PubMed Scopus Google Scholar), levels of mRNA were found in SSc fibroblasts SSc patients in the expression of mRNA SSc patients were levels of LH2b mRNA expression to mRNA expression were found in SSc fibroblasts SSc patients whereas LH1 SSc patients LH3 SSc patients and SSc patients mRNA to expression were Analysis of the collagen, deposited by fibroblasts cultured from fibrotic skin of SSc an increase in hydroxyallysine-derived cross-links, indicating that the hydroxylation of the collagen telopeptides is increased in the fibrotic skin of SSc patients SSc patients This is in with in data R. M. M. J. Dermatol. 2001; PubMed Scopus Google Scholar). is characterized by an excessive accumulation of collagen. Furthermore, a change in the collagen cross-link is found in fibrotic to collagen accumulation in fibrosis are the of collagen and the of of these is that not the the collagen and in the are it has been that the change in a in the of allysine-derived cross-links in of hydroxyallysine-derived cross-links, is a in fibrosis (8Brinckmann J. Notbohm H. Tronnier M. Acil Y. Fietzek P.P. Schmeller W. Muller P.K. Batge B. J. Invest. Dermatol. 1999; 113: 617-621Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Brinckmann J. Neess C.M. Gaber Y. Sobhi H. Notbohm H. Hunzelmann N. Fietzek P.P. Muller P.K. Risteli J. Gebker R. Scharffetter-Kochanek K. J. Investig. Dermatol. 2001; 117: 269-273Abstract Full Text Full Text PDF PubMed Google Scholar, 10Last J.A. King T.E. Nerlich A.G. Reiser K.M. Am. Rev. Respir. Dis. 1990; 141: 307-313Crossref PubMed Scopus (49) Google Scholar, 11Ricard-Blum S. Bresson-Hadni S. Vuitton D.A. Ville G. Grimaud J.A. Hepatology. 1992; 15: 599-602Crossref PubMed Scopus (78) Google Scholar, 12Ricard-Blum S. Esterre P. Grimaud J.A. Cell. Mol. Biol. 1993; 39: 723-727PubMed Google Scholar, 13Uzawa K. Marshall M.K. Katz E.P. Tanzawa H. Yeowell H.N. Yamauchi M. Biochem. Biophys. Res. Commun. 1998; 249: 652-655Crossref PubMed Scopus (34) Google Scholar, 14Bailey A.J. Bazin S. Sims T.J. Le Lous M. Nicoletis C. Delaunay A. Biochim. Biophys. Acta. 1975; 405: 412-421Crossref PubMed Scopus (228) Google Scholar, 15Bailey A.J. Light N.D. CIBA Found. Symp. 1985; 114: 80-96PubMed Google Scholar). The amount of hydroxyallysine-derived cross-links as is related to the irreversible accumulation of collagen in fibrotic tissues (10Last J.A. King T.E. Nerlich A.G. Reiser K.M. Am. Rev. Respir. Dis. 1990; 141: 307-313Crossref PubMed Scopus (49) Google Scholar, 11Ricard-Blum S. Bresson-Hadni S. Vuitton D.A. Ville G. Grimaud J.A. Hepatology. 1992; 15: 599-602Crossref PubMed Scopus (78) Google Scholar, 12Ricard-Blum S. Esterre P. Grimaud J.A. Cell. Mol. Biol. 1993; 39: 723-727PubMed Google Scholar, 14Bailey A.J. Bazin S. Sims T.J. Le Lous M. Nicoletis C. Delaunay A. Biochim. Biophys. Acta. 1975; 405: 412-421Crossref PubMed Scopus (228) Google Scholar, 15Bailey A.J. Light N.D. CIBA Found. Symp. 1985; 114: 80-96PubMed Google Scholar). This that collagen containing hydroxyallysine-derived cross-links is more difficult to degrade and therefore to the accumulation of collagen. The increased amount of in fibrosis is the of an overhydroxylation of the lysine residues within the telopeptides. Although the existence of a TLH has been B. Eyre D.R. Shao P. Am. J. Hum. Genet. 1995; 57: 1505-1508PubMed Google Scholar, R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar), the gene encoding TLH to not been identified. Biochemical analysis of the bone of BS patients revealed that the BS is to the decreased activity of TLH (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google Scholar). lysyl are LH2, and encoded by PLOD2, and PLOD3, LH1 is a helical lysyl hydroxylase (23Yeowell H.N. Walker L.C. Mol. Genet. Metab. 2000; 71: 212-224Crossref PubMed Scopus (127) Google Scholar, 24Steinmann B. Eyre D.R. Shao P. Am. J. Hum. Genet. 1995; 57: 1505-1508PubMed Google Scholar), whereas the substrate specificity of LH2 and LH3 is Here we by of mutation two missense mutations in exon 17 of PLOD2 in BS indicating that LH2 is the putative TLH. These mutations are in an showing between the lysyl LH2, and and between the lysyl of from to a that this region is for the of the lysyl in and that of LH2 in Mutation analysis of patients from a BS family showing to 17p12 (29Bank R.A. Robins S.P. Wijmenga C. Breslau-Siderius L.J. Bardoel A.F. Van Der Sluijs H.A. Pruijs H.E. Tekoppele J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1054-1058Crossref PubMed Scopus (159) Google not mutations in the or the region of This that these patients must to 17 be Bruck syndrome type I, whereas patients showing mutations in PLOD2 to the Bruck syndrome type This is genetic as we are not of BS for the into the substrate specificity of it is a telopeptide lysyl hydroxylase. the increased amount of in fibrosis is likely to be the of increased TLH we investigated the expression of LH2 in fibrotic tissues. Analysis of and LH3 mRNA expression in fibrotic skin of SSc patients revealed of the the mRNA of LH2b to was LH2a was in both and SSc fibroblasts, indicating that this splice is in skin fibroblasts and therefore no in Furthermore, we found normal mRNA levels of LOX, which is the enzyme catalyzing the formation of allysine and a for the formation of The increase in pyridinoline cross-links as found in of SSc patients is therefore not to increased aldehyde Together, these imply that LH2b can be responsible for the overhydroxylation of collagen telopeptide lysine residues in SSc to the increased formation of It is therefore to that LH2b an in the irreversible accumulation of collagen in The type of cross-links a for the of collagen (10Last J.A. King T.E. Nerlich A.G. Reiser K.M. Am. Rev. Respir. Dis. 1990; 141: 307-313Crossref PubMed Scopus (49) Google Scholar, 11Ricard-Blum S. Bresson-Hadni S. Vuitton D.A. Ville G. Grimaud J.A. Hepatology. 1992; 15: 599-602Crossref PubMed Scopus (78) Google Scholar, 12Ricard-Blum S. Esterre P. Grimaud J.A. Cell. Mol. Biol. 1993; 39: 723-727PubMed Google Scholar, 14Bailey A.J. Bazin S. Sims T.J. Le Lous M. Nicoletis C. Delaunay A. Biochim. Biophys. Acta. 1975; 405: 412-421Crossref PubMed Scopus (228) Google Scholar, 15Bailey A.J. Light N.D. CIBA Found. Symp. 1985; 114: 80-96PubMed Google Scholar). The of the formation of hydroxyallysine-derived cross-links by specifically TLH in to the formation of allysine-derived cross-links is likely to the collagen more to to a in collagen accumulation. of this is that the collagen for normal of the is not as TLH is specifically in Inhibition of LH2 is therefore an to with fibrotic LH2 a and for van for