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• W1967864208 abstract "Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1–3) separated by short non-collagenous domains (NC1–4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an α-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen α chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule. Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1–3) separated by short non-collagenous domains (NC1–4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an α-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen α chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule. Type XIII collagen is a type II transmembrane protein that is expressed in many tissues throughout development and adult life (1Sund M. Väisänen T. Kaukinen S. Ilves M. Tu H. Autio-Harmainen H. Rauvala H. Pihlajaniemi T. Matrix Biol. 2001; 20: 215-231Crossref PubMed Scopus (60) Google Scholar). It is located in focal adhesions of cultured fibroblasts and other cells and in adhesive structures of tissues such as the myotendinous junctions in muscle, intercalated discs in heart, and the cell basement membrane interphases (1Sund M. Väisänen T. Kaukinen S. Ilves M. Tu H. Autio-Harmainen H. Rauvala H. Pihlajaniemi T. Matrix Biol. 2001; 20: 215-231Crossref PubMed Scopus (60) Google Scholar, 2Hägg P. Väisänen T. Tuomisto A. Rehn M. Tu H. Huhtala P. Eskelinen S. Pihlajaniemi T. Matrix Biol. 2001; 19: 727-742Crossref PubMed Scopus (58) Google Scholar). The type XIII collagen ectodomain can bind to fibronectin, heparin, the basement membrane components nidogen-2 and perlecan, and the α1-subunit of integrin (3Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar, 5Tu H. Sasaki T. Snellman A. Göhring W. Pirilä P. Timpl R. Pihlajaniemi T. J. Biol. Chem. 2002; 277: 23092-23099Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Due to its location at the tissue and cell level and its binding properties, it has been postulated that type XIII collagen is involved in cellular adhesion and migration.Type XIII collagen α chains produced in a recombinant insect cell culture system have been shown to form homotrimers (6Snellman A. Keränen M.R. Hägg P.O. Lamberg A. Hiltunen J.K. Kivirikko K.I. Pihlajaniemi T. J. Biol. Chem. 2000; 275: 8936-8944Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The primary structure is composed of three collagenous domains (COL1 1The abbreviations used are: COL, collagenous domain; NC, non-collagenous domain; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; 4PH, prolyl 4-hydroxylase; MSRs, macrophage scavenger receptors; MES, 4-morpholineethanesulfonic acid; SRCL, scavenger receptor with C-type lectin.1The abbreviations used are: COL, collagenous domain; NC, non-collagenous domain; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; 4PH, prolyl 4-hydroxylase; MSRs, macrophage scavenger receptors; MES, 4-morpholineethanesulfonic acid; SRCL, scavenger receptor with C-type lectin. to COL3), which are flanked and interrupted by non-collagenous domains (NC1 to NC4). The short cytosolic domain and the transmembrane domain encompass about half of the NC1 domain, whereas the rest of the molecule forms the ectodomain, which is a rod of about 150 nm with two flexible hinges coinciding with the NC2 and NC3 domains (5Tu H. Sasaki T. Snellman A. Göhring W. Pirilä P. Timpl R. Pihlajaniemi T. J. Biol. Chem. 2002; 277: 23092-23099Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The primary structures of COL1, NC2, COL3, and NC4 can vary, on account of complex alternative splicing (7Juvonen M. Pihlajaniemi T. J. Biol. Chem. 1992; 267: 24693-24699Abstract Full Text PDF PubMed Google Scholar, 8Juvonen M. Sandberg M. Pihlajaniemi T. J. Biol. Chem. 1992; 267: 24700-24707Abstract Full Text PDF PubMed Google Scholar, 9Juvonen M. Pihlajaniemi T. Autio-Harmainen H. Lab. Invest. 1993; 69: 541-551PubMed Google Scholar, 10Peltonen S. Rehn M. Pihlajaniemi T. DNA Cell Biol. 1997; 16: 227-234Crossref PubMed Scopus (27) Google Scholar). It has been shown recently that extracellular sequences adjacent to the transmembrane domain are important for the association of type XIII collagen into trimeric molecules (4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar). It appears that triple helix formation proceeds in the opposite orientation than for the fibrillar collagens, i.e. from the N terminus to the C terminus. Because type XIII collagen does not contain a signal sequence, its translocation to the endoplasmic reticulum has been thought to be mediated by the transmembrane domain sequences, residues 37–59 in the mouse and 39–61 in man (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), as is known to occur with other type II transmembrane proteins (12High S. Laird V. Trends Cell Biol. 1997; 7: 206-210Abstract Full Text PDF PubMed Scopus (37) Google Scholar).By using homologous gene targeting, we have previously generated a mouse line, Col13a1 N/N, expressing modified type XIII collagen that lacks the extreme 96 N-terminal residues, including the cytosolic, transmembrane, and association domains, which are replaced by unique sequences not found in any other protein (13Kvist A.P. Latvanlehto A. Sund M. Eklund L. Väisänen T. Hägg P. Sormunen R. Komulainen J. Fässler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Analysis of tissues and cultured cells derived from homozygous Col13a1 N/N mice has shown that the altered type XIII collagen molecules are transported to roughly the correct location despite the lack of a transmembrane domain. Expression of the N-terminally altered type XIII collagen molecules results in changes in muscle integrity in the gene-targeted mice, including abnormalities in the sarcolemma-basement membrane interphase. Immunoelectron microscopy has indicated that the mutant molecules are situated in the adjacent extracellular space, whereas wild-type type XIII collagen molecules are adherent to the plasma membrane. Moreover, cells extracted from the mutant mice showed decreased adhesiveness to the basement membrane component type IV collagen.Studies with the Col13a1 N/N mice have revealed a role for the N terminus of type XIII collagen in anchoring muscle cells to the basement membrane. Nevertheless, the mutant molecules were secreted, and they may retain intact some aspects of their functional properties. This prompted us to study the molecular properties of the N-terminally altered type XIII collagen. The amount of type XIII collagen in tissues is very low, and thus insect cell expression was used to obtain sufficient protein for these studies. A series of N-terminal variants was tested for their ability to form stable disulfide-bonded type XIII collagen molecules. The data led us to search for other regions in addition to the NC1 domain that may be important for chain association and stability in type XIII collagen and other collagenous transmembrane and non-transmembrane proteins.EXPERIMENTAL PROCEDURESConstruction of Expression Vectors and Generation of Recombinant Baculoviruses—Human type XIII collagen variant del1–38 and del1–83 viruses have been described previously (6Snellman A. Keränen M.R. Hägg P.O. Lamberg A. Hiltunen J.K. Kivirikko K.I. Pihlajaniemi T. J. Biol. Chem. 2000; 275: 8936-8944Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). E-26 (14Pihlajaniemi T. Tamminen M. J. Biol. Chem. 1990; 265: 16922-16928Abstract Full Text PDF PubMed Google Scholar), a cDNA clone covering the coding sequence for type XIII collagen except for the beginning of the translation, was used as a template for generating human variant del1–61. Complementary oligonucleotides (nucleotides 660–692 in human type XIII collagen cDNA bearing a translation start codon in position 678–680, under GenBank™ data base accession number AJ293624 (4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar)) were used as primers in a mutagenesis reaction performed using a site-directed mutagenesis kit according to the manufacturer's protocol (Stratagene). The cDNA was transported to the insect cell expression vector pVL1392 (Invitrogen) by EcoRI digestion and ligation.Sequences coding for the altered N termini of mouse type XIII collagen were obtained by reverse transcription followed by amplification with PCR. Total RNA was isolated from the skeletal muscle of a mouse expressing N-terminally altered type XIII collagen and transcribed into single-stranded DNA using a type XIII collagen-specific reverse oligonucleotide primer complementary to nucleotides 1022–1039 (under GenBank™ data base accession number NM_007731 (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)) as described previously (13Kvist A.P. Latvanlehto A. Sund M. Eklund L. Väisänen T. Hägg P. Sormunen R. Komulainen J. Fässler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The long altered N-terminal sequences were amplified using a sense oligonucleotide primer corresponding to nucleotides in the loxP sequence (5′-CGGGGTACCGAATTCTGTATGCTATACGAAGTTATTAG-3′, see Ref. 13Kvist A.P. Latvanlehto A. Sund M. Eklund L. Väisänen T. Hägg P. Sormunen R. Komulainen J. Fässler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar for further explanation) and the short sequences using a primer corresponding to nucleotides 5818–5840 in the first intron (under GenBank™ data base accession number AF063666 (17Kvist A.P. Latvanlehto A. Sund M. Horelli-Kuitunen N. Rehn M. Palotie A. Beier D. Pihlajaniemi T. Matrix Biol. 1999; 18: 261-274Crossref PubMed Scopus (16) Google Scholar)). KpnI and EcoRI restriction enzyme recognition sequences were included in the 5′-end of both sense primers, and the antisense primer used in both amplifications was complementary to nucleotides 984–1003 in mouse type XIII collagen cDNA (under GenBank™ data base accession number NM_007731 (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)). The mouse type XIII collagen cDNA moXIII (689), lacking exons 15, 31, and 36 (4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar), was digested with KpnI thereby cutting off the wild-type 5′-sequences from the rest of the clone. PCR products were also digested with KpnI and linked to replace the wild-type 5′-sequences. Recombinant expression constructs XIIIN-long and XIIIN-short were generated by releasing the inserts from the Bluescript vectors by EcoRI digestion and linking them to the pVL1393 expression vector.Recombinant baculoviruses were generated by transfecting the construct DNAs together with modified Autographa californica nuclear polyhedrosis virus DNA into Spodoptera frugiperda Sf9 insect cells using the BaculoGold transfection kit (Pharmingen). The recombinant viruses were plaque-purified and amplified as described previously (18Gruenwald S. Heitz J. Baculovirus Expression Vector System: Procedures & Methods Manual. Pharmingen, San Diego1993Google Scholar).Analysis of Recombinant Proteins Produced in Insect Cells by SDS-PAGE and Immunoblotting—High Five insect cells were cultured as monolayers in TNM-FH (Sigma) insect cell medium supplemented with 10% fetal bovine serum (Bioclear) and, when infected, in serum-free Express Five medium (Invitrogen) at 27 °C. Viruses coding for the various type XIII collagen variants were used at m.o.i. 5 together with the virus coding for both subunits of human prolyl 4-hydroxylase (4PHαβ) (19Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (50) Google Scholar) at m.o.i. 1. For infection, insect cells were seeded on plates at a density of 1 × 105 cells/cm2. As a control, cells were infected with the prolyl 4-hydroxylase virus alone. Fresh ascorbate phosphate (80 μg/ml, Wako Pure Chemical Industries Ltd.) was added to the infected cells daily.The cells were detached 48 h post-infection by gently pipeting with the medium. The cells were collected by centrifuging at 340 × g for 10 min at room temperature, and the medium was supplemented with 2 mm EDTA or Complete Protease Inhibitor Mixture (Roche Applied Science) to prevent proteolytic cleavage. The cells were washed with PBS and collected as described previously. They were then homogenized in 67 mm Tris-HCl, pH 7.5, 267 mm NaCl, 0.2% Triton X-100 supplemented with Complete Protease Inhibitor Mixture and incubated for 30 min on ice. The cell lysate was centrifuged at 8000 × g for 10 min at 4 °C; the supernatant was recovered, and the precipitate was dissolved in 1% SDS. Samples of the different fractions were analyzed by denaturing SDS-PAGE under reducing or non-reducing conditions. The samples were subjected to Western blot analysis with anti-human type XIII polyclonal antibody XIII/NC3-1 (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), detected with enhanced chemiluminescence. The percentage of secreted protein was estimated by scanning densitometry using the Quantity One Quantitation software (Bio-Rad).Pepsin Digestions of Recombinant Proteins—Cell lysate supernatants not supplemented with any protease inhibitor were digested with 0.1–0.15 mg/ml pepsin (Roche Applied Science) for 2–5 min at room temperature. Samples were analyzed by SDS-PAGE followed by Western blotting with anti-human type XIII collagen antibodies XIII/NC2-55 (6Snellman A. Keränen M.R. Hägg P.O. Lamberg A. Hiltunen J.K. Kivirikko K.I. Pihlajaniemi T. J. Biol. Chem. 2000; 275: 8936-8944Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), XIII/NC3-1 (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and XIII/NC4-SO (6Snellman A. Keränen M.R. Hägg P.O. Lamberg A. Hiltunen J.K. Kivirikko K.I. Pihlajaniemi T. J. Biol. Chem. 2000; 275: 8936-8944Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and anti-mouse type XIII collagen antibody XIII/NC1-Q610 (2Hägg P. Väisänen T. Tuomisto A. Rehn M. Tu H. Huhtala P. Eskelinen S. Pihlajaniemi T. Matrix Biol. 2001; 19: 727-742Crossref PubMed Scopus (58) Google Scholar).Recombinant Protein Purification, N-terminal Sequencing, and Pepsin Digestions—High Five cells in suspension were cultured in Express Five medium in a Certomat BS 4 shaker (B. Braun Biotech) with 130 rpm horizontal agitation at 27 °C. The High Five cells were co-infected at a density of 1 × 106/ml with the virus encoding mouse XIIIN-short at m.o.i. 5 and with the virus 4PHαβ at m.o.i. 1. Once they were infected, ascorbate phosphate (80 μg/ml) was added to the culture medium daily. 500 ml of cell culture medium was separated from the cells 48 h post-infection by centrifuging at 340 × g for 10 min at room temperature, and 2 mm EDTA was added, after which the medium was further centrifuged at 40,000 × g for 45 min at 4 °C to remove the debris and viruses. The medium was then applied to a Resource S 6-ml column (Amersham Biosciences) and eluted using a gradient program on ÄKTA explorer 10 (Amersham Biosciences). The fractions were analyzed by Western blotting using the antibody XIII/NC3-1, and those containing XIIIN-short protein were concentrated to 1 ml and purified on a Sephacryl S-500 column (1.6 × 100 cm, Amersham Biosciences) using 20 mm HEPES, pH 7.0, 0.15 m NaCl. The relevant fractions were then concentrated to 0.5 μg/μl (total protein), and about 10 μg of total protein was separated on 10% SDS-PAGE gel under reducing conditions and electroblotted onto a ProBlott™ membrane (Applied Biosystems), which was stained with Serva Blue R (Serva). The authenticity of the XIIIN-short protein was confirmed by N-terminal protein sequencing using a 492 Procise™ protein sequencer (Applied Biosystems).40 μg of partly purified XIIIN-short protein was digested with 0.8 μgof pepsin (50:1 w/w) for 2 min at room temperature. The digestion products were separated by SDS-PAGE and electroblotted onto a ProBlott™ membrane for N-terminal protein sequencing analysis.For the sequencing of XIIIN-long proteins in the cell lysate, infected insect cells from a 100-ml suspension culture were harvested after 48 h of infection by centrifuging at 340 × g for 10 min at room temperature. They were then washed with 10 ml of PBS twice, homogenized with 10 ml of PBS, and incubated for 30 min on ice. The debris and insoluble molecules were removed by centrifugation at 40,000 × g for 45 min at 4 °C. The supernatant of the cell lysate was separated out in steps using a HiTrap Q 5-ml column (Amersham Biosciences), by increasing the concentration of NaCl in the PBS buffer. The fractions containing XIIIN-long were concentrated to 0.5 ml and subsequently precipitated with 75% cold ethanol for 30 min. The ethanol precipitates were dissolved in SDS-PAGE sample buffer, applied to SDS-PAGE, and electroblotted onto a ProBlott™ membrane for N-terminal protein sequencing analysis.For the sequencing of XIIIN-long proteins in medium, the medium sample described above was centrifuged at 40,000 × g for 45 min at 4 °C to remove the virus and small debris and then loaded onto a HiTrap Q 1-ml column (Amersham Biosciences) pre-equilibrated with 20 mm MES, 0.3 m NaCl, 2 mm EDTA, pH 6.0, at 0.5 ml/min, followed by step-by-step elution with the same buffer containing 0.3, 0.5, 0.8, and 1 m NaCl, respectively. All the fractions were analyzed by Western blotting using the antibody XIII/NC3-1. The fractions containing XIIIN-long protein were combined and dialyzed against 20 mm Tris, 0.15 m NaCl, 2 mm EDTA, pH 7.4, and loaded onto a Resource Q 1-ml column (Amersham Biosciences) pre-equilibrated with the same buffer at 0.5 ml/min at 4 °C. The elution was performed by means of a programmed gradient with ÄKTA Explorer 10 starting from 0.15 to 1.0 m NaCl in the 20 mm Tris, 2 mm EDTA, pH 7.4, buffer for a 20-column volume. The elution fractions containing the XIIIN-long protein were concentrated to 1 ml and further separated by Superdex 200 (Amersham Biosciences) in ÄKTA Explorer 10 with 20 mm HEPES, 0.15 m NaCl, pH 7.0. The fractions containing XIIIN-long protein were concentrated and applied to the SDS-PAGE followed by electroblotting onto a ProBlott™ membrane for N-terminal protein sequencing analysis.Amino Acid Sequence Analysis and Secondary Structure Prediction— Sequence alignment of collagen types XIII, XXIII, XXV, and XXVI was performed using the ClustalW method (20Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55190) Google Scholar) and compiled into a figure using BOXSHADE (ch.EMBnet.org). Coiled-coil predictions for collagen types XIII, XVII, XXIII, XXV, XXVI, MARCO, and EDA were made using the COILS program (version 2.1 (21Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3452) Google Scholar)). The GenBank™ data base accession numbers for collagen types XIII, XVII, XXV, XXVI, MARCO, and EDA are AJ293624, NM_000494, AF293340, AB085837, NM_006770, and NM_001399, respectively. The positions of the heptad repeats in the NC1 and NC3 domain of type XIII collagen were predicted according to the COILS program.RESULTSSecretion of Type XIII Collagen Molecules Expressed in Insect Cells—The type XIII collagen molecules synthesized by the Col13a1 N/N mice lacked the cytosolic, transmembrane, and association domains (the 96 extreme N-terminal residues encoded by exon 1) but retained the large collagenous ectodomain. Surprisingly, these molecules were located in association with adherence structures apparently secreted into the pericellular matrix, and they were correctly located in focal adhesions in cultured cells derived from these mice (13Kvist A.P. Latvanlehto A. Sund M. Eklund L. Väisänen T. Hägg P. Sormunen R. Komulainen J. Fässler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To test the effect of the altered N terminus on association and folding of the mutant α1(XIII) chains, we prepared recombinant DNA expression constructs encoding identically altered mouse α1(XIII) chains (Fig. 1), the deleted N-terminal sequences in the Col13a1 N/N mice being replaced by either 65 or 11 residues of sequences unique to the mutant α1(XIII) chains depending on which of the two potential new translation initiation sites was used. Consequently, two constructs were prepared, namely XIIIN-long and XIIIN-short, corresponding to full-length α1(XIII) chains from residue 97 onwards and preceded by the longer or shorter mutant N termini (Fig. 1A). The del1–38 variant lacks the cytosolic domain but corresponds to full-length human α1(XIII) chains in terms of chain association and folding, and this was used as a control on account of its superior expression levels relative to full-length human and mouse α chains (Fig. 1B (6Snellman A. Keränen M.R. Hägg P.O. Lamberg A. Hiltunen J.K. Kivirikko K.I. Pihlajaniemi T. J. Biol. Chem. 2000; 275: 8936-8944Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar)). In addition, constructs del1–61 and del1–83 were prepared, encoding human α1(XIII) chains lacking either the first 61 or 83 residues, respectively (Fig. 1B). In each case insect cells were infected with viruses encoding α1(XIII) and prolyl 4-hydroxylase (4PHαβ), the latter being necessary in order to obtain hydroxylated recombinant collagen chains when using insect cells as hosts (22Lamberg A. Helaakoski T. Myllyharju J. Peltonen S. Notbohm H. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1996; 271: 11988-11995Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar).Proteins were extracted from infected insect cells by homogenizing the cells in a buffer containing Triton X-100, and the remaining precipitates were solubilized in 1% SDS. The volumes of the cell and medium fractions were adjusted to correspond to the same cell number, and the samples were fractionated on denaturing SDS-PAGE gels under reducing or non-reducing conditions and analyzed by Western blotting. Because the SDS fractions contained only minimal amounts of protein, these were excluded from the final pictures, which contain only the Triton X-100 cellular fractions and medium samples for each variant (Fig. 2). It has been shown previously that about half of the del1–38 α1(XIII) chains are secreted into the medium through proteolytic cleavage by one or more furin-type proteases when cultured in medium supplemented with serum (4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar). A furin consensus sequence can be found at amino acid residues 105–108 in human type XIII collagen (under GenBank™ data base accession number CAC00688 (4Snellman A. Tu H. Väisänen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar)) and at residues 103–106 in the mouse protein (under GenBank™ data base accession number NP_031757 (11Hägg P. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)). Here we used serum-free medium, and only about 10% of the del1–38 protein was secreted (Fig. 2B, lanes 1 and 2), possibly because of a decrease in proprotease activators in the medium. Similar or slightly higher portions of the protein, namely about 50% for XIIIN-short, 15% for XIIIN-long, 40% for del1–61, and 15% for del1–83, were found to be secreted (Fig. 2B, lanes 3–10).Fig. 2Western blotting analysis of expressed recombinant proteins in cell and medium fractions under reducing or non-reducing conditions. Cells were infected with viruses coding for various type XIII collagen variants together with a virus encoding both prolyl 4-hydroxylase subunits. Medium samples were collected 48 h post-infection, and proteins were extracted from the cells with a buffer containing Triton X-100. Proteins analyzed by 10% SDS-PAGE under reducing conditions are shown in A and by 5% SDS-PAGE under non-reducing conditions are shown in B. The samples for both panels were derived from cells infected with viruses coding for del1–38 (lanes 1 and 2), XIIIN-short (lanes 3 and 4), XIIIN-long (lanes 5 and 6), del1–61 (lanes 7 and 8), and del1–83 (lanes 9 and 10) and extracted with a buffer containing Triton X-100 (lanes 1, 3, 5, 7, and 9), or else the medium proportioned to the cell number (lanes 2, 4, 6, 8, and 10). The antibody used was XIII/NC3-1. Trimers (T), dimers (D), and monomers (M) are shown by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutant XIIIN-short and XIIIN-long α1(XIII) Chains Are Processed at a Furin Site—Western blotting of XIIIN-long cell fractions revealed two bands with molecular masses of 90 and 80 kDa (Fig. 2A, lane 5), whereas the predicted molecular mass for XIIIN-long is 64 kDa. This difference is known to reflect the high imino acid content of collagens in addition to post-translational modification of the polypeptides (23Furthmayr H. Timpl R. Anal. Biochem. 1971; 41: 510-516Crossref PubMed Scopus (422) Google Scholar). Purification and sequencing of the cell fraction proteins indicated that the 90-kDa band has the N-terminal MLYEVIRSLE predicted for intact XIIIN-long (13Kvist A.P. Latvanlehto A. Sund M. Eklund L. Väisänen T. Hägg P. Sormunen R. Komulainen J. Fässler R. Pihlajaniemi T. Am. J. Pathol. 2001; 159: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), whereas the 80-kDa band corresponded to polypeptides with the N-terminal 147GQPGEKGAPG located at amino acid residue 147 in the mouse protein, and thus the latter represented a degradation product of the full-length XIIIN-l" @default.
• W1967864208 created "2016-06-24" @default.
• W1967864208 creator A5001336307 @default.
• W1967864208 creator A5036252076 @default.
• W1967864208 creator A5046138571 @default.
• W1967864208 creator A5066280928 @default.
• W1967864208 date "2003-09-01" @default.
• W1967864208 modified "2023-09-27" @default.
• W1967864208 title "Type XIII Collagen and Some Other Transmembrane Collagens Contain Two Separate Coiled-coil Motifs, Which May Function as Independent Oligomerization Domains" @default.
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