Proc. Natl. Acad. Sci. USAVol. 87, pp. 1606-1610, February 1990Biochemistry

Identification of a distinct type IV collagen a chain with restrictedkidney distribution and assignment of its gene to the locus of Xchromosome-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-terized a type IV collagen a chain, referred to as a5(IV). Fouroverlapping cDNA clones isolated contain an open readingframe for 543 amino acid residues of the carboxyl-terminal endof a collagenous domain, a 229-residue carboxyl-terminalnoncollagenous domain, and 1201 base pairs coding for a 3'untranslated region. The collagenous Gly-Xaa-Yaa repeat se-quence has five imperfections that coincide with those in thecorresponding region of the al(IV) chain. The noncollagenousdomain has 12 conserved cysteine residues and 83% and 63%sequence identity with the noncollagenous domains of theal(IV) and a2(IV) chains, respectively. The a5(IV) chain hasless sequence identity with the putative bovine a3(IV) anda4(IV) chains. Antiserum against an a5(IV) synthetic peptidestained a polypeptide chain of about 185 kDa by immunoblotanalysis and immunolocalization of the chain in human kidneywas almost completely restricted to the glomerulus. The genewas assigned to the Xq22 locus by somatic cell hybrids and insitu hybridization. This may be identical or close to the locus ofthe X chromosome-linked Alport syndrome that is believed tobe a type IV collagen disease.

Type IV collagen, the major component of basement mem-branes, consists primarily of al(IV) and a2(IV) chains (1).The complete primary structure of the al(IV) and a2(IV)chains from man (2, 3) and mouse (4, 5) and an a(IV) chainof Drosophila (6) have been reported. All these chainscontain a highly conserved carboxyl-terminal noncollage-nous domain (NC domain) of 227-231 residues and a collag-enous domain of 1398-1456 residues. Unlike fibrillar colla-gens, the type IV collagen a chains contain numerous inter-ruptions in the otherwise continuous Gly-Xaa-Yaa repeatsequence. The existence of two distinct type IV collagena3(IV) and a4(IV) chains has also been reported (7, 8), whichindicates that this protein must have several molecular com-positions. Immunological studies indicate that the major formof collagen IV containing the al(IV) and a2(IV) chains is aubiquitous basement membrane component (see ref. 1).However, this may not always be the case, as the presenceof other type IV collagen a chains in these antigens has notbeen ruled out. Evidence for region specificity, at least in thekidney, has been obtained with antibodies against the puta-tive a3(IV) chain (9).

Diseases, such as the Goodpasture syndrome and theAlport syndrome, that seem to be associated with type IVcollagen defects provide models for studies of the minorchains. The Goodpasture antigen has been proposed to be thea3(IV) NC domain (9). The Alport syndrome is an X chro-

mosome-linked disease primarily characterized by hematuriaand patchy splitting of the glomerular basement membrane(GBM) (10-12). There is evidence that, at least in certainkindreds, the pathological mechanism is an alteration or eventhe complete absence of a type IV collagen-like a chain (13,14). However, the defect in the Alport syndrome cannot bein the al(IV) or a2(IV) genes since they have been mappedto 13qter (15, 16).

MATERIALS AND METHODSIsolation and Characterization of cDNA Clones. A human

placenta cDNA library in Agtll (Clontech) was screened (3)using an oligonucleotide mixture TG(TC)CA(AG)GT(ACG-T)TG(TC)ATG (32 permutations) that codes for a type IVcollagen NC domain consensus sequence Cys-Gln-Val-Cys-Met. The filters were rehybridized with cDNA clonesHT-21 (17) and HD-4 (18), which code for the human al(IV)and a2(IV) chains, respectively. Clones that were positive forthe oligonucleotide mixture but not for the al(IV) and a2(IV)cDNAs were isolated, subcloned into M13 (19) vector, andsequenced (20) for initial characterization. For Nothern blotanalysis (21), total RNA was isolated from frozen tissuesusing an acid guanidinium thiocyanate/phenol/chloroformextraction procedure (22).

Synthetic Peptides and Preparation of Antibodies to ana5(IV)-Derived Peptide. An a5(IV) peptide Ser-Asp-Met-Phe-Ser-Lys-Pro-Gln-Ser-Glu and an al(IV) peptide Ser-Glu-Met-Phe-Lys-Lys-Pro-Thr-Pro-Ser were made. For thepreparation of antiserum, the a5(IV) peptide was coupled toovalbumin (23) and a rabbit was immunized.Immunological Methods. For blotting, normal human skin

fibroblasts were extracted with SDS buffer and electropho-resed under reducing conditions (24). The separated proteinswere transferred to a nitrocellulose filter for immunostaining.Immunohistological staining was carried out on 4-,um-thickmethanol-fixed cryosections from an adult human kidneyobtained at autopsy. Fluorescein isothiocyanate- (JanssenBiochimica) or peroxidase- (Zymed Laboratories) conju-gated anti-rabbit IgG was used for immunostaining. Peroxi-dase activity was visualized using 4-chloro-1-naphthol (25)and H202 as substrates.Chromosomal Assignment. Chromosomal assignment of

the human a5(IV) gene was carried out using a panel con-sisting of 36 human-mouse hybrids derived from 14 unrelatedhuman cell lines and 4 mouse cell lines (26, 27). Southern blotanalysis was performed with 10 ,g of HindIII-digested DNAfrom each cell line by using 32P-labeled MD-6 cDNA as probe

Abbreviations: NC, noncollagenous; GBM, glomerular basementmembrane.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(28). In situ chromosome hybridization was performed asdescribed (29, 30) using 3H-labeled MD-6 cDNA as probe.

RESULTSIsolation of cDNA Clones Coding for a5(IV), Type IV

Collagen a Chain. The strategy for the isolation of cDNAclones coding for potential type IV collagen a chains was tomake use of the fact that there are short identical amino acidsequences in the carboxyl-terminal NC domain of the humanand mouse al(IV) and a2(IV) chains (17, 18, 31, 32) as wellas in the evolutionarily distant Drosophila a(IV) chain (6). Itwas concluded that these sequences are essential for thestructure or function of the protein, and they could, there-fore, be expected to be present in all other potential a chainsof type IV collagen. One such sequence is Cys-Gln-Val-Cys-Met. A 15-mer synthetic oligonucleotide coding forthis sequence was used as probe for screening of the cDNAlibrary.Primary screening of 360,000 plaque-forming units yielded

720 duplicate positive signals. Since it could be anticipatedthat a large portion of the signals were derived from al(IV)and a2(IV) cDNA clones, the clones HT-21 (17) and HD-4(18) coding for these chains, respectively, were used torescreen the same filters under stringent hybridization andwashing conditions (21). This resulted in eight negativeclones that were subcloned into M13 vectors and sequencedfrom both ends for initial characterization. Six ofthose codedfor a unique type IV collagen-like a chain. The longest clone,MD-6 [1.4 kilobases (kb)], encoded a short part of a 3'untranslated region, a complete NC domain, and a part of thecollagenous domain (Fig. 1).The MD-6 clone was then used to rescreen the same cDNA

library and this yielded, among others, one 2.0-kb clone(PL-31) that reached 955 base pairs (bp) further upstream andtwo clones of 2.4 kb (PC-4) and 1.8 kb (PL-35) that coded forthe 3' untranslated region, the NC domain, and a part of thecollagenous domain (Fig. 1).

Nucleotide and Amino Acid Sequence of the Human a5(IV)Chain. The complete nucleotide sequencer ofthe overlappingclones for the type IV a chain we have termed a5(IV)revealed a 2316-bp open reading frame coding for 772 aminoacid residues and a 1201-bp 3' untranslated region (Fig. 2).The PC-4 cDNA clone has 1167 bp of a 3' untranslated regionsequence and a 22-nucleotide poly(A) tail that is preceded bytwo potential polyadenylylation signals, AATTAAA orAATATA (33), instead of the typical AATAAA consensussequence (Fig. 2). The PL-35 cDNA clone that has no poly(A)tail reaches 34 nucleotides further downstream of PC-4,indicating that at least two sizes of mature mRNAs exist.The cDNA-deduced amino acid sequence contains 543

residues of a collagenous Gly-Xaa-Yaa repeat sequence thathas five imperfections of 2-5 residues and a 229-residuecarboxyl-terminal NC domain. The NC domain contains 12cysteine residues. The conserved Cys-Gln-Val-Cys-Met se-quence was located at the same site as in the other knowntype IV collagen a chains (Fig. 2). Northern blot analysis withtotal RNA from human embryonic kidney and lung demon-strated that the gene codes for a transcript(s) of about 6.5 kband the signal is about three times more intense with RNAfrom kidney than from lung (Fig. 3). Using kidney total RNA,the ratio of the bands was about 40:20:1, respectively, withal(IV), a2(IV), and a5(IV) cDNA probes of similar size andspecific activity (data not shown). Therefore, the a5(IV)chain is a minor basement membrane component in wholekidney.

COLLAGENOUS DOMAIN NC-DOMAIN 3' UNTRANSLATED

5, PL-31B C M 6 k TAA

PC-4 K K T+Ak PL-35 K T*A

K H

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 kb

FIG. 1. Partial restriction map of four overlapping cDNA clonescoding for the human a5(IV) chain. The translation stop codon(TAA), two potential polyadenylylation signals (AATATA and AAT-TAAA), and a poly(A) tail are indicated. The regions of the mRNAencoded by the clones are indicated at the top. Restriction sites forBamHI (B), HinclI (Hc), HindIlI (H), and Kpn I (K) are indicated.

Comparison of the Human a5(IV) Chain with the al(IV),a2(IV), a3(IV), and a4(IV) Chains. The 772-residue car-boxyl-terminal end sequence of a5(IV) was compared withthe corresponding sequence of the human al(IV) and a2(IV)chains (Fig. 2). All the chains are remarkably similar but thea5(IV) chain is more closely related to the a 1(IV) chain thanto the a2(IV) chain. In the NC domain the sequence identitybetween the a5(IV) chain and a 1(IV) is 83% whereas it is 63%with the a2(IV) chain. The NC domain of the a5(IV) andal(IV) chains has 229 residues whereas it is 227 residues inthe a2(IV) chain. The sequence available from the collage-nous domain of the a5(IV) chain is 58% identical with thecorresponding region of the al(IV) chain but only 46%identical with that of the a2(IV) chain. All the interruptionsin the collagenous domain match in their location between theal(IV) chain and the a5(IV) chain (Fig. 2).

Short sequences around the presumable junction betweenthe collagenous domain and the NC domain of the putativebovine a3(IV) and a4(IV) chains have been reported (7, 8).Comparison of those with the corresponding region of thea5(IV) chain shows sequence identity with the a3(IV) ofonly46%, when the figure for the putative a4(IV) chain is only24%. This extensive difference cannot be explained by in-terspecies differences because, for example, the human andmurine al(IV) chains have complete sequence homology inthis region (4, 17).We, therefore, conclude that the cDNA clones described

here code for a5(IV), a distinct chain of the type IV collagena chain family.Immunoblot Analysis and Indirect Immunofluorescence

with Antisera Against an a5(IV) Chain-Derived Peptide. Totalprotein from cultured human fibroblasts was used for immu-noblot analysis. Filter strips containing the blotted proteinwere incubated with the antibodies made against the a5(IV)-chain-derived peptide. The antiserum stained a band of thesame size as the a 1(IV) chain (Fig. 4, lane B). This stain wasblocked by preincubation of the antiserum with the antigen(Fig. 4, lane C). The stain was not blocked by preincubationof the antiserum with a corresponding al(IV) peptide con-jugate (Fig. 4, lane D). Preimmune serum was completelynegative (Fig. 4, lane A). Nonspecific stain of a band aboutthe size of the a2(IV) chain and of lower molecular weightcould be seen in all samples (lanes A-D). In lane B somepossible degradation products are also visible.Immunofluorescence studies on cryosections from a hu-

man adult kidney with the a5(IV) peptide antiserum gave areaction that was highly restricted to the GBM whereas thebasement membrane of the Bowman's capsule was com-pletely negative (Fig. SA). Furthermore, little if any reactionwas seen in the tubular or vascular basement membranes. Noreaction was obtained with the preimmune serum (data notshown). In contrast, antiserum prepared against the NCdomain of type IV collagen isolated from human placentashowed a strong stain of the basement membranes of Bow-man's capsule and tubuli as well as the GBM (Fig. SB).

MThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M31115).

Biochemistry: Hostikka et al.

AAAAAAA

AAAAAATTAAA

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897938

14850947987

295999961037

44514910461087

58919710941137

73924711441187

88629611931237

103334512421287

118339512921337

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148349513921437

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178359514921536

193364515421584

208369515921634

223074416411684

23802530268028302980313032803430

Proc. Natl. Acad. Sci. USA 87 (1990)

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FIG. 2. Nucleotide sequence and the derived amino acid sequence of a5(IV) chain coding cDNA clones and comparison with the amino acidsequence of the human a1(IV) and a2(IV) chains. Rows: 1, nucleotide sequence numbered beginning from the 5' end; 2, deduced amino acidsequence of the a5(IV) chain; 3, corresponding sequence of the al(IV) chain where it differs from the a5(IV) chain; 4, sequence of the a2(IV)chain where it differs from the a5(IV) chain. The cysteine residues are circled and the interruptions in the collagenous Gly-Xaa-Yaa repeatsequence are boxed. The conserved sequence Cys-Gln-Val-Cys-Met (CQVCM) used for design of screening primers as well as the two sequencesused to make synthetic peptides specific for the a5(IV) and al(IV) chains are underlined. The translation stop codon (TAA) is indicated by anasterisk and the potential polyadenylylation signals are indicated by double underlines. The 3' end of the PC-4 cDNA with a poly(A) tail is shownbelow the 3' end of the entire nucleotide sequence. The single-letter amino acid code is used.

Chromosomal Assignment. Chromosomal location of the gation of human COLAS in the cell hybrid panel cocorre-human a5(IV) gene (COL4AS) was determined by hybrid- lated with the distribution of human chromosome X. All otherization of the 1400-bp MD-6 cDNA clone to DNA from a chromosomes segregated discordantly with COMAS. Anpanel of 36 human-mouse somatic cell hybrids. The segre- examination of cell hybrids retaining different human X-

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kb

23.1-9I4-6.6-4.3 -

2.3 -2.0 -

FIG. 3. Northern blot analysis of totalRNA from human embryonic kidney andlung tissues. RNA (10,ug) was separatedon a 1.0%o agarose gel, blotted to nitro-cellulose, and hybridized with a 1.2-kb32P-labeled fragment from the 3' untrans-lated region of the PL-35 cDNA clone.Lanes: 1, lung; 2, kidney. Migration of

1 2 size markers is indicated.

autosome translocations with various lengths of the X chro-mosome further localized COLAAS to the Xq22-26 region(cell hybrid data not presented, but are available from au-thor). In situ hybridization confirmed the localization ofCOLA5 to chromosome Xq22 (Fig. 6). No other chromo-some had counts above background.

DISCUSSIONThe present work demonstrates the existence of a previouslyunknown polypeptide chain ofbasement membrane (type IV)collagen. The amino acid sequence derived from the cDNAclones isolated in this study provided about 50% of the entireamino acid sequence from the carboxyl-terminal end of thischain, which we refer to as a5(IV). The a5(IV) chain is moreclosely related to the al(IV) chain than to the a2(IV) chain.This is evident from the exactly same size and high sequenceidentity of the NC domains and also because the locations ofall imperfections in the helical domain are conserved. Todate, there are only minor protein and sequence data avail-able from the potential a3(IV) and a4(IV) chains (7, 8). It is

_- - a1 (IV)- (t2(IV)

gm

A B

' zWA B

_

.,

C D E

FIG. 4. Immunoblot analysis with antiserum against an a5(IV)chain synthetic peptide. Total cellular protein from cultured humanskin fibroblasts was size fractionated by SDS/PAGE, transferred tonitrocellulose, and stained with antiserum. Lanes: A, preimmuneserum; B, a5(IV) chain antiserum detects a 185-kDa band; C,preincubation of the a5(IV) peptide antiserum with the antigen (100,ug/2 ml of 1:100 diluted antiserum) completely blocks the stainingreaction; D, preincubation of the a5(IV) peptide antiserum with anequal amount ofa 1(IV) peptide conjugate does not block the stainingreaction; E, two bands of the al(IV) and a2(IV) chains of 185 kDaand 170 kDa, respectively, are seen in the [3H]proline-labeled typeIV collagen from Engelbreth-Holm-Swarm (EHS) tumor electro-phoresed on the same gel.

FIG. 5. Immunolocalization of the human a5(IV) chain in humankidney. (A) The a5(IV) antiserum shows stain highly restricted to theGBM. No stain is seen in the Bowman's capsule basement membraneand little if any stain can be seen in the tubular basement membrane.No stain was obtained with the preimmune serum (data not shown).(B) For comparison, antiserum against the NC domain of humanplacental type IV collagen shows intense stain in the GBM as well asthe basement membranes of the Bowman's capsule and tubuli.(Nikon Optiphot microscope; x 14.)

evident from the present study that the sequence of thea5(IV) chain is distinct from the sequences of those chains.Antiserum to a 10-residue synthetic peptide with a se-

quence from the NC domain was made. The peptide antigenhad a 50% sequence identity with the sequence from theal(IV) chain, which was the lowest possible sequence iden-tity for a 10-peptide sequence. Immunoblot analysis againsttotal protein from human fibroblasts showed that the a5(IV)antiserum stained a band of the same size as the a 1(IV) chain(185 kDa). Since the stain could be blocked by preincubationof the antiserum with the a5(IV)-peptide-ovalbumin antigenbut not by preincubation with the a l(IV)-peptide-ovalbuminconjugate we conclude that the antiserum does not detecta 1(IV).

Interestingly, immunostaining of kidney was restricted tothe GBM, indicating a highly tissue-specific distribution ofthe a5(IV) chain. There was no apparent stain in the tubularbasement membranes or in the Bowman's capsule. Antibod-ies against the putative NC domain of the a3(IV) and a4(IV)chains have shown strong reaction in the GBM and in theBowman's capsule (9). Reactivity of our a5(IV) chain anti-bodies with these chains cannot be ruled out. The antiserumagainst human placenta type IV collagen stained the GBM,Bowman's capsule and tubular basement membranes. Theplacental antigen consists presumably mainly of a 1(IV) anda2(IV) chains but the presence of other type IV collagenchains cannot be excluded. Accordingly, the al(IV) and

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1610 Biochemistry: Hostikka et al.

We thank Juha and Leila Risteli for generously providing antibod-ies against human type IV collagen and Helena Autio-Harmainen forproviding the human kidney tissues. This work was supported in partby grants from the National Research Council for Natural Sciences,Academy of Finland, The Juselius Foundation, and the NationalInstitutes of Health (GM20454 and HD05196).

13 FIG. 6. Idiogram of chromo-21.1 s some X showing silver grain dis-21.2 s tribution using the COL4A521.3 probe. One hundred metaphases

q _gggq 23 domly examined. The distribution

24 of chromosomal silver grains iden-25 tified a region on the long arm of

chromosome X that encodes26 COL4AS. The average number of27 grains was 1.7 per metaphase and28 8.9% of the total grains were lo-

cated at Xq22 and 37.5% of thegrains on chromosome X were lo-

x cated at Xq22.

a2(IV) chains might also have various distributions in dif-ferent basement membranes of the body. To date there are noimmunohistological data on the tissue specificity of a1(IV)and a2(IV). Thus type IV collagen appears to be a consid-erably more complex protein than previously thought withvarious chain compositions, tissue specificity, and functions.The results of this study establish that the human a5(IV)

chain gene (COL4AS) is on chromosome X at band q22,which is particularly intriguing with respect to the Alportsyndrome. This disease has four X chromosome-linked phe-notypes of which at least two have been mapped to Xq22-24in three studies (10, 12). As described above, the GBM isstructurally defective and some evidence suggests that thereis an alteration or even complete absence of a type IVcollagen-like a chain (13). Kashtan et al. (14) have reportedthat an Alport (X chromosome-linked) patient who obtaineda renal allograft developed antibodies against a 26-kDa NCdomain-like component. This is the same size as that of theNC domain of the a l(IV) chain (14). It is obvious that thechain possibly missing in Alport patients is not al(IV) sinceits gene has been localized to 13q. Consequently, the 26-kDacomponent detected by the Alport antiserum could be the NCdomain of a5(IV), as it has almost exactly the same size asthat of the al(IV) chain.

Current data strongly suggest that one or even more typeIV collagen a chains are defective in the GBM of certainpatients with X chromosome-linked Alport syndrome. Thiscould be due to a mutation involving gene(s) coding for typeIV collagen a chain(s), but no type IV collagen gene haspreviously been assigned to this chromosome. SinceCOL4AS is located in the locus of the Alport syndrome, it isquite possible that it is involved in the generation of one typeof the X chromosome-linked disease. However, Southernblot analysis on DNA from one male patient with X chro-mosome-linked Alport syndrome did not reveal an abnormalpattern with several restriction enzymes (data not shown),indicating no major rearrangement-e.g., a deletion in the 3'end of the gene. There might also be another type IV collagena chain on this locus in analogy with the genes for the humanal(IV) and a2(IV) chains on 13q (34). The definite answerabout the involvement of the a 5(IV) locus or another possiblyclosely linked locus for a type IV collagen a chain in Alportsyndrome, therefore, requires further detailed studies on theentire a5(IV) gene locus in both normal individuals andAlport patients.

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