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September 1986 FEBS 4028 Volume 206, number 1 Proposed alignment of helical interruptions in the two subunits of the basement membrane (type IV) collagen Gabriel Vogeli, Elizabeth Horn+, Jane Carter* and Paul S. Kaytes Molecular Biology Research and *Biopolymer Chemistry Research, The Upjohn Company, Kalamazoo, MI49001, USA Received 1 August 1986 We have isolated a cDNA clone for part of the a2 type IV collagen (pCIV-Z 176). Deoxynucleotide sequence analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C-terminal globular domain of the a2 (IV) chain. By aligning the deduced amino acid sequence of the a2 (IV) chain with the published sequence for the al (IV) chain, we find that all interruptions in the aI (IV) chain coincide with an interruption in the a2 (IV) chain. Additonal interruptions in the a2 (IV) chain exist, however, three out of the four analysed only slightly disturb the collagen triple helix. Mouse Collagen DNA sequencing Helical interruptions 1. INTRODUCTION Basement membranes are essential histological and physiological barriers in animals and humans [1,2]. Type IV collagen that forms the scaffold of the basement membrane [3] has two cy1 (IV) and one 1x2(IV) subunit [4]. The central part of each subunit consists of a Gly-X-Y repeat, that unlike the interstitial collagens, is interrupted in the LYI (IV) collagen chain (51. To determine whether such helical interruptions also exist in the ~2 (IV) collagen chain, and to analyse their position relative to the a1 (IV) chain [5-81, we characterized a partial cDNA clone for the a2 (IV) chain. 2. MATERIALS AND METHODS The cDNA clone pCIV-2-176 was isolated from a mouse Engelbreth-Holm-Swarm (EHS) tumor cDNA library (in the PstI site of pBR322) [7] by hybridizing the cDNA library [9] at low stringency + Present address: 20879, USA Oncor, Inc., Gaithersburg, MD Basement membrane Alignment (e.g. 0.9 M NaCl, 30% formamide at 42°C for 16 h, washed in 15 mM NaCl, 1.5 mM Na citrate at 25°C) with pCIV-1-225, an (~1(IV) cDNA clone [7]. The hybridization signal from pCIV-2-176 was removed at high stringency (washing at 51 “C). Standard protocols [lo] were used to prepare, subclone and sequence the DNA. 3. RESULTS AND DISCUSSION pCIV-2-176 was isolated from a cDNA library [7] prepared from RNA isolated from the Engelbreth-Holm-Swarm (EHS) tumor, tissue that is rich in basement membrane components [l 11. We hybridized the cDNA library under nonrestrictive conditions with labelled DNA from pCIV-1-225, an (~1 (IV) cDNA clone [7]. We argued that under these conditions cDNA clones related to collagen will hybridize, and that some of the positive colonies will code for the cy2(IV) collagen chain. The identity of pCIV-2-176 as an cy2 (IV) collagen clone was established by sequence comparison (Kuhn, K. and Timpl, R., personal communication). pCIV-2- 176 also overlaps at its Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50 0 1986 Federation of European Biochemical Societies 29 Volume 206, number 1 DNA GGcGcK;Gcm a2 . al l-lmxmxmm L P . F P ~~l-mx%xA~mmm . v S .kgP I T . . 120 F P . . Asp F . ASP 1 . Asp X . X . GJu X CM G~X;GTGACATTGGR~CC~;KZ~~ZT~ A -hP A T P . V A . V F .GJuT . L P . S P . L Lys . GluArg . T . I P . L ~ys . F F . GduLys . . P P . I . I P . L kg . G3u . AspGJn . I A . F . S p . Gju . GJu . AGln. S P . LLys.GlnT . T . LGln. P T . aa X I 243 v ASP P X A 1 A al X.Sl.lP.MP.SP.LX.SP.SV.YP.SP.LP.Gd~X.~pLys.LP.L *ASP’ . F P . 1 T . M A AAmx&KNxcll-mK 369 F P . L . Glu P . S Asp . I P . PGln_GduP ,kg S . GluLys . Glu ATCEA4TMXO3Zr~TG8 a2 Asp . I I’ . V X . Glu A . L f’ . T P . P A 615 ccmxmGATaxcA~~ ASP P al . GlnLys . 03 F P . F P . P T . AspArg . F P . S Lys . AspLys . LGln. 621 . Aspkg . Glu A hsn T L P . P V . V P . GluLys . Glukg . . . Glu V - F P . L A . S P . . . Glu S Lys ~~K~~;GM~;~~I~~~L~~~~~~TA~Tw+Iu~TGT~TA~K P .GluArg. Gin . A (B) TGKXXXXGK;F a2 a2 at Skg.GluLys. T d” . Gtn a2 DNA . l-E-T T DFH T ~~A-MTATT~~;-~~~~~ p al F zm 469 a2 a1 DNA September 1986 .AP.LP.PP.SV.GM.LP.TP.G#~X.VP.IP.PGJ~.SP.LP. (a) cnv! FEBS LETTERS P A . S . P P . P Gln . Gln F . I Am. F M P P . P P P M . P P . P P . F P .,+,.SP.kpV.AP.lF.LG’..Y L P . T P . -His P . 0 I P v LYS T 747 V Glu . P Lys . kspkg W P . A P . S . A V . Aspks . P Gln . Gln P . L P . FGln. . His . PLys.AspP . P Lys . P Lys . AspGIn . . L Gln . V Lys . AspGIn . Asp L P . A L P P . WA a2 Gln. al P ONA . P PLys.AspLys.AsnGln. CCM%TKKQ3 . V L . L P . GIuLys . P Lys . GluGln . F H . Asn T . P al P . I G . S P . . S Lys . Asp . L P . P . F Gln P .AP.SM.SP.IP I T H V . GlnLys . L C P F F al Gln . DNA aX;CK a2 PGln. a1 P P v . P . P Lys. LGln P P .AspP . FN-g. . GInGIn . V P Lys . T P . P . P P/rg A P .LysA S V . L P Y AspV . PGln . P I Lys 07 P . GluP P al Pkg. v . GInGlu. F P GluP . P . Arg P . Pkp. P . . L P . PGlu . .GluI . L LYS . . P M .HisGln. P F . ~k-g 1251 .N-gG P . S .mi. P . FArg.kpGln. F Asp . 08 A P . GlnLys s I g y I I vlys g f I v ~~TG===g~+gtgw=tcggct=t~tggts=g a2 I 129 .1--g. 1317 DNA M 999 P DNA P cl-rc a2 a2 V S P . L P . H P .argsv L P . S H . P P . t p s v asphis . Glu T . P P . 1347 t (0 Fig. 1. Deoxynucleotide sequence of pCIV-2-176, a cDNA clone for the LYZ (IV) collagen chain. The DNA sequence for the cy2(IV) collagen chain is shown in the first lane (the numbering starts arbitrarily at the 5’-end of the clone), the derived amino acid sequence in the second lane. Line 3 shows the amino acid sequence for the cyr (IV) collagen chain; from position A to B part of from the human CYI(IV) sequence (amino acid no.469 to 615 (81); followed by data from the mouse [7] to C and to the end [6]. The glycines essential for the typical collagen Gly-X-Y repeat are represented by a dot; the charged amino acids are written in the three-letter code; small letters indicate the C-terminal globular domain. The interruptions of the Gly-X-Y repeat in the cy2(IV) chain are numbered. ‘Missing’ amino acids invoked to bring the Gly-X-Y repeat in phase after an interruption are marked by a dash. 30 Volume 206, number 1 FEBS LETTERS N-terminal end with a published a2 (IV) cDNA clone [ 121. The DNA sequence from pCIV-2-176 for 1317 nucleotides and the deduced amino acid sequence for 439 amino acids of the helical Gly-X-Y repeat adjacent to the C-terminal globular domain are shown in fig. 1. The border between the helical portion and the C-terminal domain is considered the nucleation point for triple helix formation in collagen [2]. Since pCIV-2-176 contains this area we tested the alignment of the amino acid sequence of the LYZ (IV) subunit with the published sequence of the crl (IV) subunit [6-81 starting with the last glycine in the last Gly-X-Y repeat of both subunits (see fig. 1). This portion of the (~2 (IV) collagen chain contains 8 interruptions, the LYI(IV) chain only four. All interruptions seen in the (~1 (IV) chain (nos 1,3,5,7) that lack complete Gly-X-Y repeat units are also present at the same relative position in the a~ (IV) chain. Three interruptions only found in the (~2 (IV) chain consist of a simple substitution for glycine in the Gly-X-Y repeat (nos 2,6,8), and one interruption (no.4) has an additional amino acid. We find that our alignment of the two subunits positions the interruptions in the cri (IV) collagen chain opposite interruptions in the LYE (IV) collagen chain. Our alignment is based upon (i) beginning the alignment at the nucleation point, and (ii) maximizing the length of the Gly-X-Y repeats between the interruptions. In addition, by using the human cri (IV) collagen sequence (fig.1 from the Nterminus to point B), we find that interruption no.1 in the cyz (IV) of the mouse coincides with an interruption in ai (IV) from human. Our alignment of the two subunits is further supported by the fact that one interruption in the LYZ(IV) chain 73 amino acids further to the 5 ‘-end, contained in a overlapping cDNA clone [ 121, is opposite an interruption in the LYI(IV) chain [8] (not shown). For the above reasons, we assume that the register we have chosen for the presentation of our data (fig. 1) is the most probable alignment. The flexibility of the type IV collagen molecule seen in the electron microscope [ 131 can be explained by our finding that helical interruptions are opposite each other. Since the major interruptions (1,3,5,7) in the Gly-X-Y repeats of the two different chains of type IV collagen are in identical September 1986 positions, we propose that the genes for the two collagen chains arose from the duplication of an ancestral gene which contained the common interruptions (1,3,5,7). We could then assume that the ancestral cyz (IV) chain underwent further mutations in the course of evolution. We find that single base pair substitutions can account for the appearance of three (nos 2,4,8) of the 4 unique interruptions in the a2 (IV) chain. In interruption no.2, Ser is substituted for Gly and in interruption nos 6 and 8, Ala is substituted for Gly. Either one of these substitutions will not affect the physical parameters of the collagen molecule substantially (Mao, B. and Vogeli, G., unpublished data from computer modelling). It is, however, also possible that the cyz (IV) chain was the ancestral gene with many interruptions that were then repaired to form an cyi (IV) subunit able to form a more stable triple helix. Further support for a common ancestral gene comes from the finding that some of the regions between the interruptions have a high homology to each other and that the C-terminal end of both chains has a high homology (Kaytes, P.S., unpublished). During the submission of this manuscript a report of clones similar to ours appeared in the literature: Schwarz et al. (1986) Eur. J. Biochem. 157, 49-56. ACKNOWLEDGEMENTS We thank Dr R. Timpl and Dr K. Kuhn for amino acid sequencing data of the cyz(IV) collagen; Boryeu Mao for advice on the 3 dimensional structure of collagen and Kathy Hiestand for help with the manuscript. The present work had been initiated by G.V. and E.H. at the Laboratory for Developmental and Molecular Biology, National Eye Institute, Bethesda, MD 20205. REFERENCES 111Laurie, G.W. and Leblond, C.P. (1983) J. Histochem. Cytochem. 31, 159-163. 121Pi% K.A. and Reddi, A.H. (1984) Extracellular Matrix Biochemistry, Elsevier, Amsterdam, New York. 131 Tim& R., Wiedemann, H., Van Delden, v., Furthmayr, H. and Kuhn, K. (1981) Eur. J. Biothem. 120, 203-211. 31 Volume 206, number 1 FEBS LETTERS [4] Tryggvason, K., Robey, P.G. and Martin, G. (1980) Biochemistry 19, 1284-1289. [5] Schuppan, D., Timpl, R. and Glanville, R.W. (1980) FEBS Lett. 115, 297-299. [6] Oberbaumer, I., Laurent, M., Schwarz, U., Sakurai, Y., Yamada, Y., Vogeli, G., Voss, X., Siebold, B., Glanville, R.W. and Kuhn, K. (1985) Eur. J. Biochem. 147, 217-224. [7] Nath, P., Laurent, M., Horn, E., Sobel, M.E., Zon, G. and Vogeli, G. (1986) Gene, in press. [8] Babel, W. and Glanville, R.W. (1984) Eur. J. Biothem. 143, 545-556. 32 September 1986 [9] Vogeli, G., Horn, E., Laurent, M. and Nath, P. (1985) Anal. Biochem. 151, 442-444. [lo] Maniatis, T., Frish, E.F. and Sambrook, J. (1982) Cold Spring Harbor Laboratory, NY. [ll] Orkin, R.W., Gehron, P., McGoodwin, E.B., Martin, G.R., Valentine, T. and Swarm, R. (1977) J. Exp. Med. 145, 204-220. [12] Kurkinen, M., Bernard, M.P., Barlow, D.P. and Chow, L.T. (1985) Nature 317, 177-179. [ 131 Hofmann, H., Voss, T. and Kuhn, K. (1984) J. Mol. Biol. 172, 325-343.