Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1606-1610, February 1990 Biochemistry Identification of a distinct type IV collagen a chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome (basement membranes/glomerulus/hereditary nephritis/Goodpasture syndrome) SIRKKA LIISA HOSTIKKA*, ROGER L. EDDYt, MARY G. BYERSt, MATTI HOYHTYA*, THOMAS B. SHOWSt, AND KARL TRYGGVASON* *Biocenter and Department of Biochemistry, University of Oulu, 90570 Oulu, Finland; and tDepartment of Human Genetics, Roswell Memorial Park Institute, New York State Department of Health, Buffalo, NY 14263 Communicated by Marilyn Gist Farquhar, November 6, 1989 ABSTRACT We have identified and extensively charac- mosome-linked disease primarily characterized by hematuria terized a type IV collagen a chain, referred to as a5(IV). Four and patchy splitting of the glomerular basement membrane overlapping cDNA clones isolated contain an open reading (GBM) (10-12). There is evidence that, at least in certain frame for 543 amino acid residues ofthe carboxyl-terminal end kindreds, the pathological mechanism is an alteration or even of a collagenous domain, a 229-residue carboxyl-terminal the complete absence of a type IV collagen-like a chain (13, noncollagenous domain, and 1201 base pairs coding for a 3' 14). However, the defect in the Alport syndrome cannot be untranslated region. The collagenous Gly-Xaa-Yaa repeat se- in the al(IV) or a2(IV) genes since they have been mapped quence has five imperfections that coincide with those in the to 13qter (15, 16). corresponding region of the al(IV) chain. The noncollagenous domain has 12 conserved cysteine residues and 83% and 63% sequence identity with the noncollagenous domains of the MATERIALS AND METHODS al(IV) and a2(IV) chains, respectively. The a5(IV) chain has Isolation and Characterization of cDNA Clones. A human less sequence identity with the putative bovine a3(IV) and placenta cDNA library in Agtll (Clontech) was screened (3) a4(IV) chains. Antiserum against an a5(IV) synthetic peptide using an oligonucleotide mixture TG(TC)CA(AG)GT(ACG- stained a polypeptide chain of about 185 kDa by immunoblot T)TG(TC)ATG (32 permutations) that codes for a type IV analysis and immunolocalization of the chain in human kidney collagen NC domain consensus sequence Cys-Gln-Val- was almost completely restricted to the glomerulus. The gene Cys-Met. The filters were rehybridized with cDNA clones was assigned to the Xq22 locus by somatic cell hybrids and in HT-21 (17) and HD-4 (18), which code for the human al(IV) situ hybridization. This may be identical or close to the locus of and a2(IV) chains, respectively. Clones that were positive for the X chromosome-linked Alport syndrome that is believed to the oligonucleotide mixture but not for the al(IV) and a2(IV) be a type IV collagen disease. cDNAs were isolated, subcloned into M13 (19) vector, and sequenced (20) for initial characterization. For Nothern blot Type IV collagen, the major component of basement mem- analysis (21), total RNA was isolated from frozen tissues branes, consists primarily of al(IV) and a2(IV) chains (1). using an acid guanidinium thiocyanate/phenol/chloroform The complete primary structure of the al(IV) and a2(IV) extraction procedure (22). chains from man (2, 3) and mouse (4, 5) and an a(IV) chain Synthetic Peptides and Preparation of Antibodies to an of Drosophila (6) have been reported. All these chains a5(IV)-Derived Peptide. An a5(IV) peptide Ser-Asp-Met- contain a highly conserved carboxyl-terminal noncollage- Phe-Ser-Lys-Pro-Gln-Ser-Glu and an al(IV) peptide Ser- nous domain (NC domain) of 227-231 residues and a collag- were made. For the enous domain of 1398-1456 residues. Unlike fibrillar colla- Glu-Met-Phe-Lys-Lys-Pro-Thr-Pro-Ser numerous inter- preparation of antiserum, the a5(IV) peptide was coupled to gens, the type IV collagen a chains contain ovalbumin (23) and a rabbit was immunized. ruptions in the otherwise continuous Gly-Xaa-Yaa repeat skin sequence. The existence of two distinct type IV collagen Immunological Methods. For blotting, normal human a3(IV) and a4(IV) chains has also been reported (7, 8), which fibroblasts were extracted with SDS buffer and electropho- indicates that this protein must have several molecular com- resed under reducing conditions (24). The separated proteins positions. Immunological studies indicate that the major form were transferred to a nitrocellulose filter for immunostaining. of collagen IV containing the al(IV) and a2(IV) chains is a Immunohistological staining was carried out on 4-,um-thick ubiquitous basement membrane component (see ref. 1). methanol-fixed cryosections from an adult human kidney However, this may not always be the case, as the presence obtained at autopsy. Fluorescein isothiocyanate- (Janssen of other type IV collagen a chains in these antigens has not Biochimica) or peroxidase- (Zymed Laboratories) conju- been ruled out. Evidence for region specificity, at least in the gated anti-rabbit IgG was used for immunostaining. Peroxi- kidney, has been obtained with antibodies against the puta- dase activity was visualized using 4-chloro-1-naphthol (25) tive a3(IV) chain (9). and H202 as substrates. Diseases, such as the Goodpasture syndrome and the Chromosomal Assignment. Chromosomal assignment of Alport syndrome, that seem to be associated with type IV the human a5(IV) gene was carried out using a panel con- collagen defects provide models for studies of the minor sisting of 36 human-mouse hybrids derived from 14 unrelated chains. The Goodpasture antigen has been proposed to be the human cell lines and 4 mouse cell lines (26, 27). Southern blot a3(IV) NC domain (9). The Alport syndrome is an X chro- analysis was performed with 10 ,g of HindIII-digested DNA from each cell line by using 32P-labeled MD-6 cDNA as probe The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: NC, noncollagenous; GBM, glomerular basement in accordance with 18 U.S.C. §1734 solely to indicate this fact. membrane. 1606 Downloaded by guest on September 26, 2021 Biochemistry: Hostikka et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1607 (28). In situ chromosome hybridization was performed as COLLAGENOUS DOMAIN NC-DOMAIN 3' UNTRANSLATED described (29, 30) using 3H-labeled MD-6 cDNA as probe. 5, PL-31 B C M 6 k TAA PC-4 K K T+A AAAAAAA RESULTS k PL-35 K T*A AAAAAATTAAA Isolation of cDNA Clones Coding for a5(IV), Type IV K H Collagen a Chain. The strategy for the isolation of cDNA 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kb clones coding for potential type IV collagen a chains was to make use of the fact that there are short identical amino acid FIG. 1. Partial restriction map of four overlapping cDNA clones sequences in the carboxyl-terminal NC domain of the human coding for the human a5(IV) chain. The translation stop codon (TAA), two potential polyadenylylation signals (AATATA and AAT- and mouse al(IV) and a2(IV) chains (17, 18, 31, 32) as well TAAA), and a poly(A) tail are indicated. The regions of the mRNA as in the evolutionarily distant Drosophila a(IV) chain (6). It encoded by the clones are indicated at the top. Restriction sites for was concluded that these sequences are essential for the BamHI (B), HinclI (Hc), HindIlI (H), and Kpn I (K) are indicated. structure or function of the protein, and they could, there- fore, be expected to be present in all other potential a chains Comparison of the Human a5(IV) Chain with the al(IV), of type IV collagen. One such sequence is Cys-Gln- a2(IV), a3(IV), and a4(IV) Chains. The 772-residue car- Val-Cys-Met. A 15-mer synthetic oligonucleotide coding for boxyl-terminal end sequence of a5(IV) was compared with this sequence was used as probe for screening of the cDNA the corresponding sequence ofthe human al(IV) and a2(IV) library. chains (Fig. 2). All the chains are remarkably similar but the Primary screening of 360,000 plaque-forming units yielded a5(IV) chain is more closely related to the a 1(IV) chain than 720 duplicate positive signals. Since it could be anticipated to the a2(IV) chain. In the NC domain the sequence identity that a large portion of the signals were derived from al(IV) between the a5(IV) chain and a 1(IV) is 83% whereas it is 63% and a2(IV) cDNA clones, the clones HT-21 (17) and HD-4 with the a2(IV) chain. The NC domain of the a5(IV) and (18) coding for these chains, respectively, were used to al(IV) chains has 229 residues whereas it is 227 residues in rescreen the same filters under stringent hybridization and the a2(IV) chain. The sequence available from the collage- washing conditions (21). This resulted in eight negative nous domain of the a5(IV) chain is 58% identical with the clones that were subcloned into M13 vectors and sequenced corresponding region of the al(IV) chain but only 46% from both ends for initial characterization. Six ofthose coded identical with that of the a2(IV) chain. All the interruptions for a unique type IV collagen-like a chain. The longest clone, in the collagenous domain match in their location between the MD-6 [1.4 kilobases (kb)], encoded a short part of a 3' al(IV) chain and the a5(IV) chain (Fig.