Properties of the Collagen Type XVII Ectodomain

Collagen XVII is a transmembrane component of hemidesmosomal cells with important functions in epithelial-basement membrane interactions. Here we report on properties of the extracellular ectodomain of collagen XVII, which harbors multiple collagenous stretches. We have recombinantly produced subdomains of the collagen XVII ectodomain in a mammalian expression system. rColXVII-A spans the entire ectodomain from δNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains δNC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C (∼3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a triple-helical fold. These results were further substantiated by electron microscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular. Thermal denaturation experiments revealed melting temperatures of 41 °C (rColXVII-A), 39 °C (rColXVII-B), and 35 °C (rColXVII-C). In summary, our data suggest that triple helix formation in the ectodomain of ColXVII occurs with an N- to C-terminal directionality. Collagen XVII is a transmembrane component of hemidesmosomal cells with important functions in epithelial-basement membrane interactions. Here we report on properties of the extracellular ectodomain of collagen XVII, which harbors multiple collagenous stretches. We have recombinantly produced subdomains of the collagen XVII ectodomain in a mammalian expression system. rColXVII-A spans the entire ectodomain from δNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains δNC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C (∼3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a triple-helical fold. These results were further substantiated by electron microscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular. Thermal denaturation experiments revealed melting temperatures of 41 °C (rColXVII-A), 39 °C (rColXVII-B), and 35 °C (rColXVII-C). In summary, our data suggest that triple helix formation in the ectodomain of ColXVII occurs with an N- to C-terminal directionality. Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen (BP180), is a transmembrane protein that is widely known as a structural component of hemidesmosomes, although structures at cell-tissue interfaces other than hemidesmosomes may also contain collagen XVII (1Jones J.C. Hopkinson S.B. Goldfinger L.E. Bioessays. 1998; 20: 488-494Crossref PubMed Scopus (184) Google Scholar, 2Borradori L. Sonnenberg A. J. Invest. Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Mutations in the collagen XVII gene,COL17A1, lead to junctional epidermolysis bullosa, a hereditary blistering skin disease with epidermal detachment from the basement membrane (3Bruckner-Tuderman L. Royce P. Steinmann B. Connective Tissue and Its Heritable Disorders: Molecular , Genetic , and Medical Aspects. John Wiley & Sons, Inc., New York2000Google Scholar). The cDNA sequence of collagen XVII encodes a type II integral transmembrane protein of 1497 amino acid residues (4Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). It consists of an intracellular domain of 466 residues, a transmembrane domain of 23 residues, and an extracellular collagenous domain of 1008 amino acids with multiple non-collagenous interruptions. The length of the individual collagenous regions varies from 14 to 242 amino acid residues (4Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Collagen XVII exists in two molecular forms,i.e. as a full-length transmembrane homotrimer of three 180-kDa α1(XVII) chains and as a 120-kDa soluble form. The latter corresponds to the extracellular domain and is presumably released from the cell surface by furin-mediated proteolytic processing (5Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner-Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In some instances, an even shorter fragment with ∼90–100 kDa has been observed (6Hirako Y. Usukura J. Uematsu J. Hashimoto T. Kitajima Y. Owaribe K. J. Biol. Chem. 1998; 273: 9711-9717Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Some information about the molecular shape of collagen XVII under physiological conditions can be deduced from rotary shadowing electron microscopy of collagen XVII from bovine cell lines or from recombinant fragments. These studies revealed asymmetric molecules with an elongated shape and a globular, ball-like structure at one end (6Hirako Y. Usukura J. Uematsu J. Hashimoto T. Kitajima Y. Owaribe K. J. Biol. Chem. 1998; 273: 9711-9717Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 7Balding S.D. Diaz L.A. Giudice G.J. Biochemistry. 1997; 36: 8821-8830Crossref PubMed Scopus (61) Google Scholar). A 90-kDa pepsin/trypsin fragment of collagen XVII in detergent extracts of keratinocytes was resistant to further trypsin digestion at physiological temperatures, therefore suggesting that it was of triple-helical structure (5Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner-Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The entire collagen XVII as well as the C15 collagenous domain could be recombinantly produced in protease-resistant conformation, apparently not requiring propeptides (7Balding S.D. Diaz L.A. Giudice G.J. Biochemistry. 1997; 36: 8821-8830Crossref PubMed Scopus (61) Google Scholar). Triple helix formation of fiber-forming collagens was defined as a two-step process initiated by annealing and aggregation of the C-propeptides followed by C- to N-terminal folding of the three α-chains (8Olsen B.R. Hoffmann H. Prockop D.J. Arch. Biochem. Biophys. 1976; 175: 341-350Crossref PubMed Scopus (52) Google Scholar). A higher content of hydroxyproline residues in the C-terminal region of the amino acid sequence of fibrillar collagens has been considered as evidence that the C terminus is the locus of initiation of triple helix formation (8Olsen B.R. Hoffmann H. Prockop D.J. Arch. Biochem. Biophys. 1976; 175: 341-350Crossref PubMed Scopus (52) Google Scholar). Opposite to this, there is also experimental evidence that C-propeptides can be substituted for by other peptide sequences or can be deleted without apparent harm for helix formation (9Bulleid N.J. Dalley J.A. Lees J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (87) Google Scholar, 10Lees J.F. Tasab M. Bulleid N.J. EMBO J. 1997; 16: 908-916Crossref PubMed Scopus (125) Google Scholar, 11McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar). In fibril-associated collagens with interrupted triple helices, C-propeptides are completely missing, and even if nonhelical ends at the C terminus are deleted in recombinantly produced collagenous molecules, as shown for collagen XII, correct triple helix formation takes place (12Mazzorana M. Giry-Lozinguez C. van der Rest M. Matrix Biol. 1995; 14: 583-588Crossref PubMed Scopus (14) Google Scholar). The present study was designed to address the role of subdomains of collagen XVII ectodomain in triple helix formation and stability, in particular to delineate information on the directionality of the assembly process. With a variety of recombinant polypeptides expressed in a mammalian system, we obtained structural information by circular dichroism studies, protease sensitivity assays, and electron microscope studies that strongly suggest triple helix formation from the N to the C terminus of collagen XVII. Human cDNA coding for collagen XVII (4Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar) was used as the template for amplification by the polymerase chain reaction with appropriate primers. The numbering of nucleotides in cDNAs are according to Giudice et al. (Ref. 4Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar, GenBankTM accession number M91669), and the deduced protein sequence was counted from the methionine start codon (position 36 in M91669). All fragments were digested by appropriate restriction enzymes and were cloned into an episomal expression vector pCEP-Pu containing the signal peptide sequence of BM-40 and a puromycin resistance gene (13Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). Cloning of inserts into pCEP-Pu via a NheI restriction site resulted in secreted polypeptides with four additional N-terminal amino acid residues (APLA) preceding the expressed sequence (13Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). Sequences and correct in-frame insertions of all constructs were verified by DNA sequencing (Medigenomix). An expression plasmid for rColXVII-A corresponding to the extracellular domain of collagen XVII (amino acid residues 527–1497) was constructed by amplification of the cDNA with the sense primer 5′-TTCGCTAGCTATGGCACCCGCGGCGGGAGCAGAC-3′ (nucleotides 1684 to 1707) and the antisense primer 5′-ACGCGTCGACTCACGGCTTGACAGCAATACTTCTTC-3′ (nucleotides 4574 to 4596). The NheI-SalI fragment of the product was subcloned into pCEP-Pu, resulting in plasmid pCEP-rColXVII-A. This expression plasmid generates a 971-amino acid residue protein with the N terminus within the NC16a domain and the C terminus at the cognate end of the ectodomain. The expression plasmid for rColXVII-B (amino acid residues 527–1482) was constructed by amplification of the cDNA with sense primer 5′-TTCGCTAGCTATGGCACCCGCGGCGGGAGCAGAC-3′ (nucleotides 1684 to 1707) and antisense primer 5′-ACGCGTCGACTCATTGGTCACCTTTGTCTCCTTTTTCTC-3′ (nucleotides 4526 to 4551). The NheI-SalI-restricted fragment from the amplification product was ligated into pCEP-Pu, resulting in plasmid pCEP-rColXVII-B. This plasmid encodes 956 amino acid residues and is identical to rColXVII-A except for a 15-amino acid deletion (NC1 domain) at the C-terminal end. To prepare the expression plasmid for rColXVII-C (amino acid residues 527–808), the cDNA was amplified with sense primer 5′-TTCGCTAGCTGAGGAGGTGAGGAAGCTG-3′ (nucleotides 1573 to 1590) and the antisense primer 5′-ACGCGTCGACTCAGATCTTGCCTGGAG-3′ (nucleotides 2516 to 2529). The NheI-SalI fragment from this product was ligated into pCEP-Pu, resulting in plasmid pCEP-rColXVII-C. This plasmid encodes a 282-amino acid residue polypeptide comprising 40 residues of the NC16a domain (δNC16a) and the C15 domain. The expression plasmid for rColXVII-D (amino acid residues 1188–1497) was generated by cDNA amplification using the sense primer 5′-TTCGCTAGCTCCAGGCAATGTGTGGTCCAGCATC-3′ (nucleotides 3670 to 3693) and the antisense primer 5′-ACGCGTCGACTCACGGCTTGACAGCAATACTTCTTC-3′ (nucleotides 4574 to 4596). The NheI-SalI fragment was ligated into pCEP-Pu, resulting in plasmid pCEP-rColXVII-D. This plasmid encodes a 310-amino acid polypeptide spanning from the NC1 to the NC6 domain. Human embryonic kidney 293-EBNA cells (Invitrogen) were cultivated in Dulbecco's modified Eagle's medium (Life Technologies) containing 10% fetal calf serum, 0.25 mg/ml G418 (Calbiochem), 0.1 mg/ml penicillin/streptomycin (Biochrom KG), and 2 mm l-glutamine (Biochrom KG). The pCEP-rColXVII plasmids (25 μg) were separately transfected into 293-EBNA cells (one million cells/10 cm2 culture dish) using a calcium phosphate precipitation method (14Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). After a selection with 0.5 μg/ml puromycin (Calbiochem), the transfected cells were grown to confluence, washed twice with phosphate-buffered saline, and switched to serum-free medium containing 50 μg/ml ascorbic acid (Sigma), freshly prepared. The media were collected every 24 h, cooled, centrifuged to remove cellular debris, supplemented with 1 mm protease inhibitor Pefablock (Roth), and frozen at -80 °C until further use. The serum-free media (2–3 liters) containing recombinant fragments of rColXVII-A and rColXVII-B were concentrated to ∼80 ml by ultrafiltration and dialyzed against 20 mmTris-HCl, pH 8.8, at 4 °C. The material was passed over an anion exchange column (HiTrapQ, 5 ml; Amersham Pharmacia Biotech) equilibrated in the same buffer and subsequently eluted with a linear 0–0.4 m NaCl gradient. Fractions containing the recombinant fragments were concentrated by ultrafiltration to ∼0.5 ml and passed over a Superose 6 column (30 ml; Amersham Pharmacia Biotech) equilibrated in 50 mm Tris-HCl, pH 8.6, 150 mmNaCl at a flow rate of 0.3 ml/min. Fractions containing recombinant fragments were detected by SDS gel electrophoresis and Western blotting. The serum-free cell culture media (2–3 liters) containing recombinant fragments of rColXVII-C and rColXVII-D were concentrated to ∼70 ml, dialyzed against 50 mm sodium acetate, pH 4.8, at 4 °C, passed over a cation exchange column (HiTrapS; 5 ml; Amersham Pharmacia Biotech) equilibrated in the same buffer, and eluted with a linear 0–0.4 m NaCl gradient. Fractions containing the recombinant fragments were concentrated to ∼0.5 ml and passed over a Superose 12 column (30 ml; Amersham Pharmacia Biotech) equilibrated in 50 mm sodium acetate, pH 4.8, 200 mm NaCl. Fractions were collected, and recombinant fragments were identified as described above. SDS gel electrophoresis (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and Western blot analysis (16Giudice G.J. Emery D.J. Zelickson B.D. Anhalt G.J. Liu Z. Diaz L.A. J. Immunol. 1993; 151: 5742-5750PubMed Google Scholar) were performed according to standard procedures. 6% polyacrylamide gels were used for rColXVII-A and -B, and 10% polyacrylamide gels were used for rColXVII-C and -D. Polyclonal rabbit antiserum NC16a was used 1:1000 diluted for rColXVII-A, -B, and -C (17Schumann H. Baetge J. Tasanen K. Wojnarowska F. Schäcke H. Zillikens D. Bruckner-Tuderman L. Am. J. Pathol. 2000; 156: 685-695Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and polyclonal chicken antiserum Col17ecto-1 (5Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner-Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) was used 1:20-diluted for rColXVII-D. The antisera were a generous gift of Dr. L. Bruckner-Tuderman. Goat-anti rabbit (1:1000; Dako) or goat anti-chicken (1:100; Dako) antibodies conjugated to horseradish peroxidase were used as secondary antibodies. Purified recombinant rColXVII polypeptides were adjusted to a concentration of 20 μg/ml and dialyzed against 0.05% acetic acid. Far UV circular dichroism spectra (190–260 nm) were recorded in a 1-cm quartz cuvette on a Jasco J-715 A spectropolarimeter equipped with a temperature controller. The molar ellipticities were calculated on the basis of a mean residue molar mass of 96 g/mol. Thermal transition curves were recorded at a fixed wavelength (221 nm) by raising the temperature linearly at a rate of 30 °C/h using a Gilford temperature programmer. No deviation from the melting profiles were observed with extended temperature gradients. The degree of triple helicity at various temperatures was calculated by setting the 221-nm signal at 20 °C to 1 (maximal amount of triple helicity) and at 45 °C to zero (completely denatured collagen). rColXVII polypeptides were dialyzed against a solution of 50% glycerol in 0.05% acetic acid for 16 h at 4 °C. Samples were sprayed onto freshly cleaved mica using an air brush. The droplets on the mica were dried at room temperature at 10−6 mm Hg for 12 h in a vacuum coater (Edwards 306). The dried specimens were rotary-shadowed with platinum using an electron gun positioned at 6° to the mica surface and then coated with a film of carbon generated by an electron gun positioned at 90° to the mica surface. The replica were floated on distilled water and collected on formvar-coated grids. The replicas were examined on a Zeiss 109 transmission electron microscope. Length and diameter of molecules and aggregates were determined with the Scion Image program (Scion Image Corporation). For amino acid analyses, purified recombinant rColXVII polypeptides were hydrolyzed with 6 n HCl for 24 h at 110 °C. Samples were analyzed on an amino acid analyzer (Biochrom 20, Amersham Pharmacia Biotech) using ninhydrin for post-column color development. For determination of glucosylgalactosylhydroxylysine and galactosylhydroxylysine samples, up to 1 mg of purified rColXVII-C was hydrolyzed in 1 ml of 2 n KOH for 24 h at 110 °C. After hydrolysis, 100 μl of glacial acetic acid and 150 μl of 70% perchloric acid were added, mixed, and centrifuged for 10 min at 14,000 rpm. The supernatant was decanted and lyophilized. Lyophilized samples were re-dissolved in 1 ml of water and passed over a CF1 (Whatman) column to remove the majority of amino acids. Eluates were lyophilized and analyzed on an amino acid analyzer (Biochrom 20, Amersham Pharmacia Biotech). Purified native or pepsin-digested rColXVII-C and -D was subjected to SDS gel electrophoresis and transferred onto a Mini Pro Blott membrane (Applied Biosystems). Protein bands were visualized by Coomassie Blue staining and identified by comparison with immunoblotted material. Relevant protein bands were excised and loaded directly onto a Procise 494 protein sequencer (Applied Biosystems) for N-terminal sequencing. For assessment of the domain structure and stability of collagen XVII, purified recombinant rColXVII polypeptides were subjected to treatment by various enzymes. Collagenase treatment was used to release collagenous peptides, and pepsin treatment was used to remove protease-sensitive non-collagenous regions. Trypsin digestion was used to determine the melting temperature of the rColXVII fragments A and C. Digestion of purified polypeptides with highly purified bacterial collagenase (Sigma) was performed with 40 units/ml enzyme in 0.2m NH4HCO3, freshly prepared, at 37 °C for 2 h (18Fietzek P.P. Rexrodt F.W. Wendt P. Stark M. Kühn K. Eur. J. Biochem. 1972; 30: 163-168Crossref PubMed Scopus (76) Google Scholar). For pepsin digestion, 100 μl of rColXVII polypeptides (∼25 μg) were acidified by dialysis against 0.05% acetic acid and incubated with 1 μg/ml pepsin (Roche Diagnostics) at 4 °C for 24 h (19Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). After neutralization with saturated Tris solution, samples were separated by SDS gel electrophoresis followed by Western blotting using anti-NC16a and anti Col17ecto-1 antisera. For testing the triple-helical conformation with trypsin (20Bruckner P. Prockop D.J. Anal. Biochem. 1981; 110: 360-368Crossref PubMed Scopus (231) Google Scholar), the purified samples (∼20 μg/ml) were dialyzed against 100 mm Tris-HCl, pH 7.4, 0.4 m NaCl and preheated for 5 min at each desired temperature between 20 and 46 °C (2 °C steps). An aliquot of 10 μl was removed, cooled quickly to 20 °C, and treated with 10 μl of trypsin (1 mg/ml; Sigma) for 2 min. Reactions were stopped by adding 10 μl of soybean trypsin inhibitor (5 mg/ml; Sigma) to each individual sample. The incubated samples were analyzed by SDS gel electrophoresis followed by Western blotting with specific antibodies. Intensities of the immunoblotted bands were quantified with Gel-Pro-Analyzer (Media Cybernetics). To investigate triple-helix formation and stability of the collagen type XVII ectodomain, we have produced several recombinant subfragments of the ectodomain in mammalian 293 cells. These subdomains included the full-length ectodomain rColXVII-A (positions 527–1497), a C-terminal truncated form thereof, rColXVII-B (positions 527–1482), the largest collagenous stretch, C15 (positions 527–808), and a short C-terminal subdomain rColXVII-D (positions 1188–1497) spanning the NC6 to the NC1 domain. These recombinant fragments are schematically summarized in Fig. 1. Each of the constructs were designed with a heterologous signal peptide to enable secretion into the cell culture medium and proper post-translational modifications. The recombinant cell clones produced each subdomain in quantities of 1–2 μg of protein/ml medium/day. Ion exchange chromatography followed by gel filtration chromatography resulted in highly purified recombinant subdomains with apparent molecular masses of 101 kDa (rColXVII-A), 96 kDa (rColXVII-B), 30 kDa (rColXVII-C), and 33 kDa (rColXVII-D), which corresponded well with the calculated values (Fig. 2). Interestingly, the collagenous domain fragment rColXVII-C migrates as a broad band, similar to what has been published recently for a corresponding construct (21Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J. Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). No multimers have been observed after electrophoresis by SDS gel electrophoresis (Fig. 2). N-terminal sequencing of rColXVII-C, which N-terminally starts at the same position as rColXVII-A and -B, demonstrated the expected N-terminal sequence (APLAMA) but also some minor N-terminal truncations of 2–5 amino acid residues (TableI). Sequencing of the C-terminal construct rColXVII-D resulted in a sequence (APGNVWSSIS) that was three amino acid residues shorter than expected (Table I). These results indicate that the sequence APLA, which represents an artificial sequence due to the expression strategy (see “Experimental Procedures”), is not stable in the recombinant constructs. The authentic collagen type XVII sequences, however, were stable.Figure 2Purification of recombinant extracellular subdomains of human collagen type XVII. rColXVII polypeptides were purified, separated by SDS-gel electrophoresis, and stained with Coomassie Blue. A and B, rColXVII-A and rColXVII-B, respectively; control medium from nontransfected 293-EBNA cells (lane 1), protein pattern after HiTrapQ anion exchange chromatography (lane 2), and Superose 6 gel filtration chromatography (lane 3). C and D, rColXVII-C and rColXVII-D, respectively; control medium from nontransfected 293-EBNA cells (lane 1), protein pattern following HiTrapS cation exchange chromatography (lane 2), and Superose 12 gel filtration chromatography (lane 3). The relatively broad protein band in panel C (lane 3) is probably due to heterogeneity in glycosylation or in the amino acid sequence at the N-terminal end. Positions of globular marker proteins are indicated in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IN-terminal sequence analysis of rColXVII-C and -DDerived from:Amino acid sequencecDNA of rColXVII-CA ↓ A P L A M A P A A G A D L D K I G L H S D S Q E E L W M F V R K K L M M E Q E N G N L R G S P G P K... 527rColXVII-C(1) A P A A G A D L D K........ Major sequences (1–2)(2) A P L A M A..........................(3) L A M A P A...................... Minor sequences (3–5)(4) A M A P A A....................(5) M A P A......................rColXVII-C, pepsin-digested ....E Q E N G N L R G S.......cDNA of rColXVII-D A ↓ A P L A P G N V W S S I S V E D L S1188rColXVII-DA P G N V W S S I S...........The sequences derived from the cDNA are compared with results from N-terminal sequencing of constructs rCoIXVII-C and -D or with pepsin-degraded rColXVII-C. ↓ indicates the cleavage site between the signal peptide and the mature polypeptide. The amino acid residues APLA at the N terminus of each construct are a result of the cloning strategy. The first authentic amino acid residues from the α1(XVII) polypeptide and their corresponding positions are indicated in bold face. Gly-X-Y repeats are underlined. Open table in a new tab The sequences derived from the cDNA are compared with results from N-terminal sequencing of constructs rCoIXVII-C and -D or with pepsin-degraded rColXVII-C. ↓ indicates the cleavage site between the signal peptide and the mature polypeptide. The amino acid residues APLA at the N terminus of each construct are a result of the cloning strategy. The first authentic amino acid residues from the α1(XVII) polypeptide and their corresponding positions are indicated in bold face. Gly-X-Y repeats are underlined. The rColXVII-A and rColXVII-C polypeptide were further analyzed by amino acid analysis. Within the limits of error, the amino acid compositions determined correlated well with the expected compositions calculated from the cDNA (Table II). For rColXVII-A, about 30% of the total prolyl and 34% of the total lysyl residues were hydroxylated. For rColXVII-C, about 29% of the total prolyl and 36% of the total lysyl residues were hydroxylated (Table II). Some of these residues in rColXVII-C were also further modified by the attachment of a small but significant number of mono- and disaccharides (0.01 monosaccharide and 0.17 disaccharide per molecule; Table II).Table IIAmino acid and glycosylation analysis of rColXVII-A and -CResiduerColXVII-A residues/moleculerColXVII-C residues/moleculeDeterminedCalculated (cDNA)DeterminedCalculated (cDNA)HypHyp36.7Not possible14.3Not possibleAsx54.435 (Asp) 11 (Asn)13.310 (Asp) 2 (Asn)Thr31.3265.65Ser99.59911.011Glx77.840 (Glu) 38 (Gln)30.216 (Glu) 12 (Gln)Pro86.517935.155Gly257.822984.284Ala56.24812.914Cys0.000.00Val36.3359.19Met15.2237.711Ile27.8275.35Leu63.36116.115Tyr16.0241.60Phe21.0183.42His19.5124.54Hyl11.7Not possible5.5Not possibleLys22.7279.717Arg37.14213.313Hyp/Pro0.4Not possible0.4Not possibleHyl/Lys0.5Not possible0.5Not possibleTrpNot determined2Not determined1MonosaccharideNot determinedNot possible0.01Not possibleDisaccharideNot determinedNot possible0.17Not possible Open table in a new tab Limited enzymatic digestion with pepsin converted rColXVII-A into a ∼30-kDa fragment via an intermediate protein of the apparent molecular mass of 58 kDa (Fig.3, panel A, lane 2), whereas digestion with bacterial collagenase completely degraded this recombinant ectodomain under the experimental conditions. Similar data were obtained for the C-terminally truncated rColXVII-B (Fig. 3, panel B). The 30-kDa pepsin fragments corresponded to the size and the immunoreactivity of rColXVII-C (collagen XVII domain C15), indicating that this domain adopts a stable triple-helical conformation in rColXVII-A and -B and, thus, resists degradation by pepsin. Recombinant rColXVII-C itself was also resistant to limited degradation with pepsin, whereby a slightly (∼ 3 kDa) faster electrophoretic mobility of the pepsin-treated material was observed (Fig. 3, panel C). N-terminal sequencing of the pepsin-treated rColXVII-C revealed a N-terminally truncation of 32 amino acid residues, corresponding to parts of the non-collagenous δNC16a domain (Table I). Accordingly, the Gly-X-Y repeats of rColXVII-C were not degraded by pepsin. These results suggest a triple-helical conformation for rColXVII-C and indicate that more C-terminally located domains are not required for triple helix formation of domain C15. Treatment of the C-terminal rColXVII-D with pepsin resulted in a complete degradation, indicating the absence of a stabilizing triple-helical conformation (Fig. 3, panel D). Controls with bacterial collagenase demonstrated complete degradation of all recombinant polypeptides (Fig. 3, lanes 3). To obtain information of the molecular shape, the recombinant rColXVII subdomains were analyzed by electron microscopy after rotary shadowing (Fig. 4). The full-length ectodomain rColXVII-A displayed a long extended shape with a globular portion on one end (Fig. 4 A). Measurement of the lengths of individual particles revealed 3 groups of about 60–70 nm, 130–140 nm, and 240–250 nm. A length of 130–140 nm corresponds well with expected values for the ectodomain (22Hirako Y. Usukura J. Nishizawa Y. Owaribe K. J. Biol. Chem. 1996; 271: 13739-13745Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). A length of 240–250 nm may represent dimers that occur through lateral alignment, whereas shorter fragments may derive from proteolytic cleavage of the polypeptide. The globular domain at one end is about 11–12 nm in diameter. The widths of t