Expression of Type XXIII Collagen mRNA and Protein
2006; Elsevier BV; Volume: 281; Issue: 30 Linguagem: Inglês
10.1074/jbc.m604131200
ISSN1083-351X
AutoresManuel Koch, Guido Veit, Sigmar Stricker, Pinaki Bhatt, Stefanie Kutsch, Peihong Zhou, Elina Reinders, Rita A. Hahn, Rich Song, Robert E. Burgeson, Donald R. Gerecke, Stefan Mundlos, Marion K. Gordon,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoCollagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanα1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the α1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this. Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanα1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the α1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this. Tissues use specific sets of collagens, often synthesized simultaneously, to achieve and maintain particular functional properties. Most collagens are secreted and assembled within the extracellular environment; however, a growing subclass of collagens are transmembranous and inserted into the plasma membrane in a type II orientation to extend their extracellular collagenous domains from the cell surface. The subclass of transmembranous collagens currently includes types XIII, XVII, XXIII, and XXV, summarized in a recent review (1Franzke C.W. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2005; 280: 4005-4008Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The group has also been referred to as the MACITs for membrane-associated collagens with interrupted triple helices (2Pihlajaniemi T. Rehn M. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 225-262Crossref PubMed Scopus (78) Google Scholar). Type XIII collagen, the first member of the group identified (3Pihlajaniemi T. Myllyla R. Seyer J. Kurkinen M. Prockop D.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 940-944Crossref PubMed Scopus (33) Google Scholar), has an important function in muscle tissue. Engineered genetic mutations in the Col13a1 gene in one case causes cardiovascular defects (4Sund M. Ylonen R. Tuomisto A. Sormunen R. Tahkola J. Kvist A.P. Kontusaari S. Autio-Harmainen H. Pihlajaniemi T. EMBO J. 2001; 20: 5153-5164Crossref PubMed Scopus (31) Google Scholar) and in another causes abnormal skeletal muscle myofibers with progressive myopathy that is worsened by exercise (5Kvist A.P. Latvanlehto A. Sund M. Eklund L. Vaisanen T. Hagg P. Sormunen R. Komulainen J. Fassler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Type XIII collagen mediates cell attachment through integrin α1β1 but does not interact through another common collagen receptor, α2β1 (6Nykvist P. Tu H. Ivaska J. Kapyla J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The second transmembranous collagen to be identified was type XVII (7Giudice G.J. Emery D.J. Diaz L.A. J. Investig. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar), which was known for many years as bullous pemphigoid antigen 2 and BP180 prior to the elucidation of its collagenous nature. Being a component of the hemidesmosome (8Diaz L.A. Ratrie III, H. Saunders W.S. Futamura S. Squiquera H.L. Anhalt G.J. Giudice G.J. J. Clin. Investig. 1990; 86: 1088-1094Crossref PubMed Scopus (314) Google Scholar), type XVII collagen provides structural integrity to the cornea (9Jonkman M.F. de Jong M.C. Heeres K. Pas H.H. van der Meer J.B. Owaribe K. Martinez de Velasco A.M. Niessen C.M. Sonnenberg A. J. Clin. Investig. 1995; 95: 1345-1352Crossref PubMed Scopus (152) Google Scholar, 10Fujikawa L.S. Foster C.S. Gipson I.K. Colvin R.B. J. Cell Biol. 1984; 98: 128-138Crossref PubMed Scopus (155) Google Scholar, 11Gordon M.K. Fitch J.M. Foley J.W. Gerecke D.R. Linsenmayer C. Birk D.E. Linsenmayer T.F. Investig. Ophthalmol. Vis. Sci. 1997; 38: 153-166PubMed Google Scholar) and skin (12Gipson I.K. Spurr-Michaud S. Tisdale A. Keough M. Investig. Ophthalmol. Vis. Sci. 1989; 30: 425-434PubMed Google Scholar), clearly demonstrated by mutations in the COL17A1 gene that cause epidermolysis bullosa (13McGrath J.A. Gatalica B. Christiano A.M. Li K. Owaribe K. McMillan J.R. Eady R.A. Uitto J. Nat. Genet. 1995; 11: 83-86Crossref PubMed Scopus (326) Google Scholar, 14Tasanen K. Floeth M. Schumann H. Bruckner-Tuderman L. J. Investig. Dermatol. 2000; 115: 207-212Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Our laboratories added collagen XXIII to the family of transmembranous collagens by identifying a human EST 3The abbreviations used are: EST, expressed sequence tag; Col, collagenous; NC, non-collagenous; dbEST, database of ESTs; FACIT, fibril-associated collagen with interrupted triple helices; HEK, human embryonic kidney; EBNA, Epstein-Barr nuclear antigen 1; RACE, rapid amplification of cDNA ends; RT, reverse transcription; TBS, Tris-buffered saline; E, embryonic day. 3The abbreviations used are: EST, expressed sequence tag; Col, collagenous; NC, non-collagenous; dbEST, database of ESTs; FACIT, fibril-associated collagen with interrupted triple helices; HEK, human embryonic kidney; EBNA, Epstein-Barr nuclear antigen 1; RACE, rapid amplification of cDNA ends; RT, reverse transcription; TBS, Tris-buffered saline; E, embryonic day.and using its sequence to clone a fragment of chicken collagen XXIII cDNA to examine its expression in cornea (15Gordon M.K. Gerecke D.R. Hahn R.A. Bhatt P. Goyal M. Koch M. Investig. Ophthalmol. Vis. Sci. 2000; 41: S752Google Scholar). We also cloned the full-length mouse collagen XXIII cDNA sequence and deposited it into GenBank™ (accession number AF410792). Cloning and expression of the rat and human α1(XXIII) cDNAs revealed evidence that (i) the molecule is shed from the cell surface by selective proteolysis suggesting an involvement of furin and (ii) that the expression is up-regulated with metastatic potential in prostate cancer cell lines (16Banyard J. Bao L. Zetter B.R. J. Biol. Chem. 2003; 278: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). For collagen XXV, the newest member of the group, mRNA could be specifically detected in neurons and has been identified as a component of the senile plaques characteristic of Alzheimer disease (17Hashimoto T. Wakabayashi T. Watanabe A. Kowa H. Hosoda R. Nakamura A. Kanazawa I. Arai T. Takio K. Mann D.M. Iwatsubo T. EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (174) Google Scholar). Here we present the strategies for obtaining mouse, chicken, and human α1(XXIII) collagen cDNAs. The sequences and domain structures of the orthologs were compared as were the distribution and abundance of the mRNA in mature and developing tissues. The protein was examined in tissues using polyclonal antibodies generated in guinea pig and rabbit against the ectodomain of mouse collagen XXIII. The antibodies revealed that some tissues contain mostly the full-length transmembranous form, whereas in other tissues the shed ectodomain is the major form of collagen XXIII. Identification and Cloning of α1(XXIII) Collagen cDNAs—Stretches of amino acid sequences, derived from specific domains in the FACIT collagen types, were used as queries to perform BLAST searches (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) to pick up related, but unique, collagen cDNAs in the human dbEST (19Boguski M.S. Lowe T.M. Tolstoshev C.M. Nat. Genet. 1993; 4: 332-333Crossref PubMed Scopus (1135) Google Scholar). A unique clone (GenBank™ accession number W22262) was identified that encodes a portion of a triple helical domain and 18 amino acid residues of a non-collagenous domain with strong resemblance to type XIII collagen. The clone was obtained, completely sequenced, and assigned the next available number in the collagen family, α1(XXIII) (see supplemental material). In total, the composite full-length human α1(XXIII) collagen cDNA is 3,060 nucleotides and was assigned GenBank™ accession number AY898961. The human sequence was used to search mouse ESTs, and clone AW323570 was identified. After extending the cDNA sequence by 5′-RACE using mouse lung cDNA, an overlap was found with a 5′-end EST (GenBank™ accession number AW494383). The primary structure of the entire mouse α1(XXIII) collagen mRNA was verified by PCR and sequence analysis and was submitted to GenBank™ under accession number AF410792. To obtain the initial chick cDNA, primers derived from nearly identical regions in the human and mouse sequences were used to amplify 13-day chick embryo corneal cDNA. A small chick α1(XXIII) collagen cDNA of ∼100 bp was obtained and reported in the 2000 ARVO meeting abstracts issue of the journal Investigative Ophthalmology and Visual Science (15Gordon M.K. Gerecke D.R. Hahn R.A. Bhatt P. Goyal M. Koch M. Investig. Ophthalmol. Vis. Sci. 2000; 41: S752Google Scholar). The complete chick α1(XXIII) collagen cDNA was deposited in GenBank™ under accession number AY876377 (for cloning see supplemental material). The ExPASy Sim program from the Search Launcher at the Baylor College of Medicine Human Genome Sequencing Center website (found at searchlauncher.bcm.tmc.edu/) was used to align polypeptide chains. For the comparison of the longest version of the human α1(XXIII) collagen polypeptide (accession number AY158895), human type XIII collagen (accession number AJ293624), and human type XXV collagen (accession number NM_198721), placement of amino acid residues was biased to align conserved exons in each collagen type in an effort to reflect areas of conserved gene structure (see supplemental material). To accomplish this, the exon/intron structure of each gene was determined using GenBank™ tools. Tissues—Human placenta, lung, and kidney total RNA were purchased from Clontech. Human cornea was from Lions Eye Bank of Oregon (Portland, OR), and human amnion was from Marie France Champliaud-Steiner (formerly of Cutaneous Biology Research Center, Massachusetts General Hospital). All tissues were immediately frozen in liquid nitrogen until RNA isolation, which was accomplished using TRIzol™ (Invitrogen) following the manufacturer's instructions. On-column DNase digestion was performed on the RNA as outlined in appendix D instructions in the RNeasy™ minikit handbook from Qiagen. Human donor corneal epithelial, stromal, and endothelial cDNAs were kindly provided by Mitch Watsky, Sally Twining, and Kim Vaughn. Human immortalized corneal epithelial cells (20Araki-Sasaki K. Ohashi Y. Sasabe T. Hayashi K. Watanabe H. Tano Y. Handa H. Investig. Ophthalmol. Vis. Sci. 1995; 36: 614-621PubMed Google Scholar) (a gift from Dr. Fu-Shin Yu with permission of Dr. K. Araki-Sasaki), lung and esophageal cell lines A549 and HET-1A (a gift from Drs. C. S. Yang and L. Chen), esophageal cell line SEG-1 (from Drs. C. S. Yang and L. Chen with permission of Dr. David Beer), and the SKGT4 cell line (also from Drs. C. S. Yang, Luke Chen, and Xiaochun Xu with permission of Dr. David Schrump) were grown in culture, then scraped from plates, and used for RNA isolation. Mouse cornea, lung, skin, and sterna were dissected for RNA isolation; bone and tendon were purchased from Pel-Freeze Biologicals. RNA from human cell lines and all mouse tissues was isolated using Qiagen RNeasy kits. For chick tissue, corneas were dissected from 7-, 9-, 11-, 13-, and 15-day embryos. With the exception of the 7-day corneas, epithelia were removed from the stromal/endothelial layer with EDTA and dispase, and RNA was isolated as described previously (21Gordon M.K. Foley J.W. Lisenmayer T.F. Fitch J.M. Dev. Dyn. 1996; 206: 49-58Crossref PubMed Scopus (55) Google Scholar). For all tissues collected, RNA was quantified by absorbance measured at 260 nm. PCR, RT-PCR, and Relative RT-PCR—Routine 30-cycle amplifications were as described previously (21Gordon M.K. Foley J.W. Lisenmayer T.F. Fitch J.M. Dev. Dyn. 1996; 206: 49-58Crossref PubMed Scopus (55) Google Scholar). RACE reactions were as performed previously (22Koch M. Olson P.F. Albus A. Jin W. Hunter D.D. Brunken W.J. Burgeson R.E. Champliaud M.F. J. Cell Biol. 1999; 145: 605-618Crossref PubMed Scopus (214) Google Scholar) with modifications as described by Koch et al. (23Koch M. Laub F. Zhou P. Hahn R.A. Tanaka S. Burgeson R.E. Gerecke D.R. Ramirez F. Gordon M.K. J. Biol. Chem. 2003; 278: 43236-43244Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). With the exception of the human corneal epithelial, stromal, and endothelial mRNA, all other human relative RT-PCRs were performed in the linear range using the non-radioactive method described by Koch et al. (23Koch M. Laub F. Zhou P. Hahn R.A. Tanaka S. Burgeson R.E. Gerecke D.R. Ramirez F. Gordon M.K. J. Biol. Chem. 2003; 278: 43236-43244Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The human primers were AACATTCAAGAGTATGCCCACC and CACACAGTGATCTCCATCTACACGG (944-bp product). Products were run on ethidium-containing agarose gels and photographed with an Eagle Eye II digital camera. Relative RT-PCR to quantitate mRNA abundance in mouse and chick tissues as well as in human corneal epithelium, stroma, and endothelium was done incorporating radioactive nucleotide into the product as described previously (23Koch M. Laub F. Zhou P. Hahn R.A. Tanaka S. Burgeson R.E. Gerecke D.R. Ramirez F. Gordon M.K. J. Biol. Chem. 2003; 278: 43236-43244Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Koch M. Foley J.E. Hahn R. Zhou P. Burgeson R.E. Gerecke D.R. Gordon M.K. J. Biol. Chem. 2001; 276: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Human collagen XXIII primers were TAACATTCAAGAGTATGCCCACC and AGGTTCTTACAGGGCAGTA (240-bp product). Mouse collagen XXIII primers were GACCTGGAGAACCAGGACTCGATG and CATCCAAACGGATCTGTACAGGTC (247-bp product). Chick collagen XXIII primers were GGAGAAATGGGCTTATCGGGT and CGGGTAAGCCAATGAGCCCT (215-bp product). PCR was performed for 29 cycles in the linear amplification range for fragments. Bands were cut from gels, and radioactivity was counted. The collagen XXIII signal was normalized by dividing it by radioactivity counts of the attenuated 18 S product (Ambion primer to Competimer ratio, 1:20) or, for chick, by the radioactivity counts of the glyceraldehyde-3-phosphate dehydrogenase product. The resulting normalizations are called "adjusted cpm." Because all primers were tested for optimal amplification capability and samples for each species were amplified using one master mixture and run on the same gel, the histograms show the relative differences between the amplified products. Whole Mount and Tissue Section in Situ Hybridization—Two probes were generated by RT-PCR (Clontech) using the following primer pairs: forward CCCAAGTTCAGCACGCTTC and reverse T7-CCATGAATGGAGCAGGAGCTAG and forward TCCAAGGTCCCAAGGGCCTG and reverse T7CTGTCCAGCCTCACCTGAGCC. The whole mount in situ hybridizations was done as described previously (25Schwabe G.C. Trepczik B. Suring K. Brieske N. Tucker A.S. Sharpe P.T. Minami Y. Mundlos S. Dev. Dyn. 2004; 229: 400-410Crossref PubMed Scopus (95) Google Scholar), and section in situ hybridizations were performed with digoxigeninlabeled riboprobes using a semiautomated TECAN Genesis robot platform applying the Genepaint system (26Visel A. Thaller C. Eichele G. Nucleic Acids Res. 2004; 32: D552-D556Crossref PubMed Google Scholar). Both probes stained equivalent structures. Recombinant Expression of α1(XXIII) Collagen in Human Embryonic Kidney (HEK) 293-EBNA Cells—The full-length α1(XXIII) collagen cDNA could not be amplified in one piece so an AvrII site was incorporated without altering the amino acid sequence at positions 45-49. The 5′-fragment was amplified using a KpnI sequence linked to CTGAGTCTGAGCGGGCGGCTAGG as the sense primer and an AvrII sequence linked to GGTCCCGGGTAGCC as the antisense primer. The 3′-fragment was amplified with forward primer GGGACC linked to TCTGGACGGC through an AvrII sequence and, as reverse primer, an XhoI sequence linked to CTTATGCCAGCAACCAGGCACAGG. PCRs were done on mouse lung cDNA in 6% Me2SO using Long Expand DNA polymerase (Roche Diagnostics). The products were ligated (Rapid DNA ligation kit, Roche Diagnostics) into a modified pCEP-Pu vector carrying a 3′ FLAG and His6 tag. The ectodomain expression plasmid was generated by inserting the SPARC (secreted protein, acidic and rich in cysteine) signal peptide coding sequence (27Mason I.J. Taylor A. Williams J.G. Sage H. Hogan B.L. EMBO J. 1986; 5: 1465-1472Crossref PubMed Scopus (241) Google Scholar) between the HindIII and NheI sites in pCEP-4 (provided by Ernst Poeschl) and then inserting the collagen XXIII ectodomain amplified from mouse lung cDNA. The sense primer was an NheI sequence linked to ACAGGCGGCCGCCCTGCATGGCC, and the antisense primer was an XhoI sequence linked to CTTATGCCAGCAACCAGGCACAGG. Cloned and expanded products were sequenced to verify their identities. Transfection experiments and protein purification were done as described previously (22Koch M. Olson P.F. Albus A. Jin W. Hunter D.D. Brunken W.J. Burgeson R.E. Champliaud M.F. J. Cell Biol. 1999; 145: 605-618Crossref PubMed Scopus (214) Google Scholar). The vectors containing the collagen XXIII cDNAs were introduced into HEK 293-EBNA cells (Invitrogen) by transfection with the reagent FuGENE 6 (Roche Applied Science) according to the manufacturer's recommendations. The cells were selected with puromycin (1.25 μg/ml), stably transfected HEK 293-EBNA cells were pseudo-subcloned, and the highest protein-producing clones were expanded. Use of Collagen XXIII Ectodomain for Antibody Production—Antibodies against mouse collagen XXIII were produced as described previously (28Veit G. Kobbe B. Keene D.R. Paulsson M. Koch M. Wagener R. J. Biol. Chem. 2006; 281: 3494-3504Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Briefly for large scale protein production, the ectodomain-transfected cells were transferred to serum free Dulbecco's modified Eagle's medium/F-12 with Glutamax™ (Invitrogen) supplemented with 250 μml-ascorbic acid and 450 μml-ascorbic acid 2-phosphate. The collected cell culture supernatant was supplemented with 1 mm phenylmethylsulfonyl fluoride (Sigma) and, after filtration, was passed over a gelatin-Sepharose column (GE Healthcare) before being applied to a TALON metal affinity resin column (BD Biosciences). The recombinant protein was eluted stepwise with Tris-buffered saline (TBS; 20 mm Tris-HCl, pH 8.0, 150 mm NaCl) containing 40-250 mm imidazole. After dialysis against TBS the protein was used for immunization of two rabbits and two guinea pigs following standard procedures. The antisera collected from animals weeks later were purified by affinity chromatography on a column with antigen coupled to CNBr-activated Sepharose (GE Healthcare). The specific antibodies were eluted with 150 mm NaCl, 0.1 m triethylamine, pH 11.5, and the eluate was neutralized with 1 m Tris-HCl, pH 6.8. Solid Phase Assay—The specificity of the antibodies against collagen XXIII was confirmed in solid phase assays. The ectodomains of collagens XIII and XXV and the unrelated His-tagged protein unc5h2 were expressed and purified using the same method as used for collagen XXIII ectodomain production described above. The purified proteins were diluted in TBS, pH 7.4, and 10 μg/ml (500 ng/well) were coated overnight at 4 °C onto 96-well plates (Nunc Maxisorb). After washing with TBS, nonspecific binding sites were blocked with 5% skim milk powder in TBS for 2 h at room temperature. Serial dilutions of the affinity-purified anti-collagen XXIII antibodies in blocking buffer (1:300-1:100,000) were added to wells and incubated for 1.5 h. Bound primary antibodies were detected with secondary antibody that was either swine antirabbit horseradish peroxidase-coupled IgG (DakoCytomation) or rabbit anti-guinea pig horseradish peroxidase-coupled IgG (Sigma). For enzymatic reaction, 50 μl/well 0.25 mm tetramethylbenzidine and 0.005% (v/v) H2O2 in 0.1 m sodium acetate, pH 6.0 were incubated for 10 min. The reaction was stopped with 50 μl/well 2.5 m H2SO4, and the absorbance was measured at 450 nm using a microplate reader (Labsystems Multiscan MS). For analysis, comparably treated wells, but without addition of primary antibody, were used to tare measurements from antibody-treated wells. Protein Isolation from Tissues and Western Blot Analyses—Tissues were dissected from adult C57BL/6J mice and immediately frozen in liquid nitrogen. For extraction the tissues (200 mg/experiment) were thawed on ice and weighed, and 5 volumes (ml/g of wet tissue) of chilled extraction buffer (TBS, 1% Nonidet P-40, 2 mm EDTA, and proteinase inhibitor mixture (Complete, Roche Applied Science)) were added before homogenization using a Polytron tissue homogenizer (Kinematica). Insoluble material was subsequently removed by centrifugation. Western blots of tissue extracts gave high background; therefore, immunoprecipitation was done with antibody generated in one species (e.g. guinea pig) followed by Western blotting the immunoprecipitate and detecting it with antibody generated in the other species (e.g. rabbit). For immunoprecipitation, clarified tissue extracts were preincubated with 50 μl/ml gelatin-Sepharose resin for 1 h at 4°C to remove proteins that nonspecifically bind to Sepharose. The tissue extracts were then incubated overnight with 5 μg/ml polyclonal guinea pig anti-collagen XXIII ectodomain antibody covalently coupled to CNBr-activated Sepharose. Following the removal of unbound material, the immobilized antibody-antigen complexes were split, and one part was subjected to collagenase digestion (see below) whereas the other part was treated identically but without the addition of collagenase. Antibody-bound collagen XXIII was eluted from the Sepharose using the high pH elution method described above, and the eluates were concentrated in a SpeedVac concentrator (Savant). After reduction the immunoprecipitates were separated by 10% SDS-PAGE and subsequently blotted on nitrocellulose membranes using standard procedures. The concentrated collagen XXIII was detected using the rabbit polyclonal anti-collagen XXIII ectodomain antibody followed by incubation with an horseradish peroxidase-coupled swine anti-rabbit IgG (DakoCytomation). Luminescence was visualized using the ECL Plus™ Western blotting detection reagents (GE Healthcare). Collagenase Digestion—To confirm the specific detection of collagen XXIII in extracts from tissue homogenates, the antibody-precipitated protein was subjected to collagenase digestion as described previously (29Koch M. Schulze J. Hansen U. Ashwodt T. Keene D.R. Brunken W.J. Burgeson R.E. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2004; 279: 22514-22521Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The incubation with 100 units/ml highly purified bacterial collagenase (CLSPA, Worthington Biochemicals) was carried out in 250 μl of TBS containing 5 mm CaCl2 and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride (Roche Applied Science) for 4 h at 37 °C. The reaction was stopped by adding EDTA to a final concentration of 20 mm. Immunohistochemistry—Immunohistochemistry was performed on frozen OCT-embedded sections of embryonic mouse tissues preincubated in ice-cold methanol for 2 min, blocked for 1 h with 5% normal goat serum in phosphate-buffered saline containing 0.2% Tween 20, and incubated with the guinea pig collagen XXIII primary antibody overnight at 4 °C. This was followed by Cy™3-conjugated goat antiguinea pig IgG (DakoCytomation). Alexa 488-conjugated goat antirabbit IgG (Molecular Probes) was used as secondary antibody for costaining rabbit collagen XXIII polyclonal antibody or detecting laminin 5 polyclonal antibody. Stained sections were analyzed, and pictures were taken with a confocal laserscanning microscope (Leica TCS SL) using two lasers in parallel with the excitation wavelengths 488 nm for Alexa488 and 543 nm for Cy3. α1(XXIII) Collagen cDNAs—A BLAST search (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) of the dbEST (19Boguski M.S. Lowe T.M. Tolstoshev C.M. Nat. Genet. 1993; 4: 332-333Crossref PubMed Scopus (1135) Google Scholar) with triple helical sequences of FACIT collagens yielded a novel human EST clone (GenBank™ accession number W22262), which was purchased and sequenced. 5′-RACE extended the clone, and further data base searching revealed the 5′-end. The composite full-length human cDNA (GenBank™ accession number AY898961) encodes a unique collagen of 503 amino acid residues that strongly resembled type XIII collagen. It was assigned the next available number in the collagen family at the time, α1(XXIII). A BLAST search with the human sequence identified a mouse IMAGE clone (accession number AW323570), which was sequenced and extended by RACE. The mouse α1(XXIII) cDNA sequence encodes 533 amino acid residues and was deposited in GenBank™ under the accession number AF410792. Degenerate nucleotide primer pairs, designed from stretches of identical amino acids in mouse and human, were used to amplify a chick corneal cDNA, yielding an ∼100-bp product (15Gordon M.K. Gerecke D.R. Hahn R.A. Bhatt P. Goyal M. Koch M. Investig. Ophthalmol. Vis. Sci. 2000; 41: S752Google Scholar). BLAST searching of chicken ESTs yielded a partial Gallus cDNA (BU270831) (30Boardman P.E. Sanz-Ezquerro J. Overton I.M. Burt D.W. Bosch E. Fong W.T. Tickle C. Brown W.R. Wilson S.A. Hubbard S.J. Curr. Biol. 2002; 12: 1965-1969Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), which was used to identify overlapping chicken genomic clones in the data base. The composite in silico derived cDNA was then amplified and sequenced from chick corneal mRNA, and the sequence was deposited in GenBank™ under accession number AY876377. The polypeptides encoded by the mouse, chick, and human cDNAs are shown in Fig. 1 along with the sequence of the rat (16Banyard J. Bao L. Zetter B.R. J. Biol. Chem. 2003; 278: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The cDNAs define three triple helical collagenous (Col) domains and four non-collagenous (NC) domains within the polypeptide numbered from the amino to the carboxyl end to make the designations consistent with the closest relatives in the collagen family (types XIII and XXV). The mouse and chick α1(XXIII) collagen NC1 domains are slightly shorter than the human 120-residue NC1 domain (Fig. 1). In all species, the amino termini do not have characteristics of signal peptides but instead have stretches of hydrophobic residues typical for membrane-spanning regions (31Sonnhammer E.L. von Heijne G. Krogh A. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar). The short amino-terminal cytoplasmic stretches of the orthologous molecules are quite different in length and amino acid composition. The Col 1 domain of mouse, rat, and the previously reported human (16Banyard J. Bao L. Zetter B.R. J. Biol. Chem. 2003; 278: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) α1(XXIII) collagen is 189 amino acid residues in length. In all the mammalian cDNAs, the Col 1 domain contains one Gly-X-Y triplet in which the glycine is exchanged by a serine or asparagine. The chicken collagen XXIII lacks this triplet and thus has a perfect Gly-X-Y triplet structure in Col 1 domain. The newly obtained human cDNA encodes a 150-instead of a 189-amino acid residue Col 1 domain. This difference may reflect alternative splicing of collagen XXIII mRNA, a feature prevalent in the related collagen XIII mRNA (32Peltonen S. Rehn M. Pihlajaniemi T. DNA Cell Biol. 1997; 16: 227-234Crossref PubMed Scopus (27) Google Scholar, 33Tikka L. Pihlajaniemi T. Henttu P. Prockop D.J. Tryggvason K. Proc. Natl. Acad. Sci. U. S. 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