THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, , No. 18, Issue of June 25. pp. 12475-12481,1992 Printed in U. S . A.

Complete Amino Acid Sequence of the Human a5(IV) Collagen Chain and Identification of a Single-base Mutation in Exon 23 Converting Glycine 521 in the Collagenous Domain to Cysteine in an Alport Syndrome Patient*

(Received for publication, January 21, 1992)

Jing ZhouS, Jens Michael Hertz& Anu LeinonenS, and Karl TryggvasonS From the SBiocenter and Department of Biochemistry, University of Oulu, SF-90570 Oulu, Finland and the §institute of Human Genetics, Aarhus University, DK-8000 Aarhus C, Denmark

We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal pep- tide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa- Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous do- main. The calculated molecular weight of the mature a5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome re- vealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G + T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by al- lele-specific hybridization analyses.

Basement membranes are ubiquitous sheet-like extracellu- lar structures separating organ cells from the interstitial connective tissue (1). The basement membranes serve many important functions in uiuo such as cell differentiation, cell adhesion, and tissue regeneration. The basement membrane of renal glomeruli (GBM)’ is a unique type of basement membrane forming a single, well-defined layer located be- tween the endothelial cells and the epithelial podocytes. The GBM has been the focus of intensive research because it is believed to function as the actual renal filtration barrier for macromolecules. The molecular structure and pathology of

* This work was supported in part by grants from the Academy of Finland, Sigrid Juselius Foundation, Finland’s Cancer Institute (to K. T.), Helen and Ejnar Bjdrnow’s Foundation (tp J. M. H.), and Nyreforeningens Forskningsfond (to J. M. H.). The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: GBM, basement membrane of renal glomeruli; NC, noncollagenous; PCR, polymerase chain reaction; bp, base pair(s); RFLP, restriction fragment length polymorphism; ESRD, end-stage renal disease.

the GBM has also gained the attention of investigators be- cause it is primarily affected in inherited kidney diseases such as the Alport and congenital nephrotic syndromes and in some acquired kidney diseases such as diabetic nephropathy, Goodpasture syndrome, and other nephritides. Recently, X chromosome-linked Alport syndrome was shown to be caused by mutations in the COL4A5 collagen gene encoding the highly GBM-specific a5 chain of type IV collagen (2,3). This disease, which characteristically leads to end-stage renal dis- ease in affected males, is initially characterized by hematuria, GBM disruption, and frequently also hearing loss and ocular lesions (4, 5).

A type IV collagen network forms the structural skeleton of basement membranes (1). It is a complex trimeric molecule composed of at least five genetically distinct polypeptide chains; al(IV), a2(IV), a3(IV), a4(IV), and a5(IV). Three chains assemble into different isoforms of triple-helical mol- ecules, the most abundant combination being [al(IV)]z a2(IV). As yet, little is known about the composition of molecules containing the minor chains a3(IV), a4(IV), and a5(IV). The complete sequence of the al(1V) and a2(IV) chains of human and mouse are known (6-8,35) and also that of an a(IV) collagen chain from Drosophila (9). Partial se- quences of the human and bovine a3(IV) collagen chains and the bovine a4(IV) chain have been reported (10-14) and also an extensive portion of the human a5(IV) chain sequence (15, 16). The type IV collagen a chains have a homologous globular carboxyl-terminal noncollagenous (NC) domain and a collag- enous domain with a frequently interrupted Gly-Xaa-Yaa repeat sequence. The presence of a glycine in every third position is essential for the maintenance of a triple-helical conformation. Extracellularly, the type IV collagen molecules assemble into a network structure by end-to-end assembly, through disulfide cross-links between two molecules at the NC domains and four molecules at the amino termini (1). This network is further strengthened by lateral assembly of molecules (17). The genes for al(1V) and a2(IV) collagen have been located to chromosome 13 (18-20), the genes for the a3(IV) and a4(IV) chains to chromosome 2 (12),’ and the gene for the a5(IV) chain gene to the q22 region of chromo- some X (15). The major type IV collagen a1 and a 2 chains appear to be present in most if not all basement membranes as opposed to the a3, a4, and a5 chains that have a restricted tissue distribution. Importantly, the aS(1V) chain is located in the kidney solely in the GBM (15).

Six different mutations have been reported in the COLAAS collagen gene in six unrelated kindreds with Alport syndrome

S. Reeders and M. Mariyama, personal communication.

12475

12476 Human cu5(IV) Collagen Chain

(2, 3, 22). The mutations which both involve large deletions and point mutations in the gene can be considered to be sufficient to produce malfunctional a5(IV) chains that can weaken the GBM structure and result in the Alport pheno- type.

The complete primary structure of the a5(IV) collagen chain as well as its gene are important for studies on type IV collagen in general, but also for further elucidation of the molecular genetics and phenotypes of X chromosome-linked Alport syndrome. In the present study we have generated cDNA clones providing the entire amino acid sequence of the human a5(IV) collagen chain, which was shown to contain 1685 residues. Additionally, one point mutation was identified within the sequence of one of the new clones in a kindred with Alport syndrome. The mutation causing a collagenous domain glycine 521 substitution to cysteine was shown to be located in exon 23, which was isolated and sequenced together with its adjacent intron regions.

EXPERIMENTAL PROCEDURES

Isolation and Characterization of cDNA Clones and Generation of a Primer-extended cDNA Library-A cDNA library made from human fibrosarcoma cell (HT-1080) mRNA (Clontech) was screened with a ""P-labeled 5'-end fragment from the previously described PL-31 3'- end (u5(IV) chain cDNA clone (see Ref. 15 and Fig. 2) using standard protocols (23). Several new overlapping clones, including HT-11 and HT-14, reaching upstream from PL-31, were isolated. However, in- tensive screening did not yield clones coding for the most 5'-end part of the mRNA. In order to obtain such clones, a primer-extended cDNA library was made using poly(A) RNA isolated from human kidney tissue using the guanidine thiocyanate method (23). An oli- gonucleotide, named CTE, containing the sequence 5"TGGTCCT- GGCAGTGATGACATAATTATACT-3' from the 5' end of HT-14 (nucleotides 680-711, see Fig. 3) was synthesized and used for primer extension. The primer-extended cDNA library was made using cDNA synthesis and package kits (Promega) according to the manufacturer's instructions. This library was screened with a 0.69-kb EcoRI cDNA fragment (HT-14-1) from the 5' end of HT-14, and a 0.72-kb cDNA clone (JZ-4) extending 0.45-kb upstream from HT-14 was obtained. The cDNA clones were sequenced from both strands by the dideoxy method (24) using T7 DNA polymerase (Sequenase, United States Biochemical).

Alport Syndrome Pedigree Material-A pedigree of Danish kindred A21 is shown in Fig. 1. The proband, Iv4, is a 33-year-old male with a juvenile form of Alport syndrome accompanied by sensorineural deafness. Microscopic hematuria and proteinuria were detected inci- dentally a t the age of 8, and the disease progressed to terminal renal failure at the age of 29. Renal biopsy carried out a t the age of 20 revealed occasional foci of chronic inflammatory cells and a few foam cells in the interstitium by light microscopy. Electron microscopic examination of the biopsy specimen was not undertaken. The proband has been transplanted once with a kidney from his father, and this kidney is still well functioning. A progressive sensorineural hearing defect started at the age of 20, and at the age of 30, a bilateral hightone loss of 50 db was revealed by audiometric testing. Lenticonus has not been detected by ophthalmological examination. His maternal grandfather, 111, died at 37 years of age of chronic kidney disease. He also had a hearing defect. The grandfather's mother, three brothers, and one sister also died of chronic kidney disease in their early thirties. The proband's maternal aunt, IIL, had proteinuria from early childhood and microscopic hematuria was found on several occasions. At the age of 41, she was admitted to hospital because of hypertension due to chronic renal failure with creatinine clearance of 35-42 ml/min and proteinuria 3 glliter. The renal failure progressed and at the age of 50 she had a cerebral hemorrhagic insult resulting in left-sided hemiparesis and she died at 53 in terminal uremia. Sensorineural deafness was found at the age of 30 on audiometric testing. In HIh, the mother of the proband, proteinuria and micro- scopic hematuria started at the age of 6, and the renal disease progressed to terminal uremia at the age of 41. She has been trans- planted twice; the first graft was rejected within a few months but the second graft functioned well. Her hearing was reduced, but audiometric testing was not performed. She died at 60 years of age.

In IV,, proteinuria and microscopic hematuria were detected at the

n I

c 5.1 kb

c 1.35 kb + 1.25 kb

FIG. 1. Top, pedigree of kindred A21. Obligate female carriers and affected females are shown as open circles with a dot. Unaffected females are shown as open circles and unaffected males as open boxes. Affected males are shown as black boxes. The proband Iv4 is indicated by an arrow. Bottom, Southern blot of HindIII-digested genomic DNA from IVI, VI, IV,, IV,, and Iv4 chains from kindred A21 in lanes 1-5 numbered from left to right. Lanes 6 and 7 are indicated by d and correspond to normal male controls. The filter was hybridized with "2P-labeled insert from the n5(IV) collagen cDNA clone HT-14-2. The approximate lengths of the fragments in kb are given on the right. The proband Iv4 is missing a 1.35-kb band and shows a variant 1.25-kb band. His female-cousin IV, is heterozygous having the normal 1.35-kb band as well as the variant 1.25-kb band.

age of 18 during pregnancy. The proteinuria and hematuria have continued after delivery, and she is now 45 years old. Her blood pressure is 155/95 mm Hg and serum creatinine is 94 pmollliter (reference interval, 40-90 pmollliter). There are no hearingproblems, but audiometric testing has not been performed recently. V I is a 27- year-old female and healthy without hematuria or proteinuria. Serum creatinine is 79 pmollliter, and the blood pressure is 145/85 mm Hg. IV, is 33 years old and healthy. Proteinuria and hematuria were not detected during childhood. She was hospitalized when 31 years old because of gastroenteritis, and during that stay microscopic hematuria and proteinuria were detected. By control a few months later she was completely well. Serum creatinine is 61 pmollliter, and the blood pressure is 150/95 mm Hg. IVa is 28 years old, and, apart from a severe bilateral congenital sensorineural hearing defect, she is healthy without hematuria or proteinuria. Serum creatinine is 76 pmollliter, and the blood pressure is 125/75 mm Hg. Only IV,, IVp, IVa, Iv4, and VI were available for study.

Southern Hybridization Analysis-Genomic DNA was extracted from lymphocytes from the five members of family A21 indicated in Fig. 1, as well as from two normal male controls. Samples of 8 pg were digested to completion with Hind111 (Boehringer) and electro- phoresed on 0.7% agarose gels. The DNA was blotted to Hybond-N membranes (Amersham), and the filters were hybridized with a5(IV) chain cDNA inserts after 32P labeling of the probes by oligonucleotide priming (25).

Isolation of Genomic Clone LA26 Containing Exon 23-As a part of

Human a5(IV) Collagen Chain 12477

our on-going work to elucidate the entire structure of the human cu5(IV) collagen gene (COUA5), genomic clones were isolated and characterized. A human X chromosome-specific library in Charon 35 (ATCC 57750) was screened with the 32P-labeled HT-14 cDNA clone containing the sequence encoding the central and amino-terminal parts of the a5(IV) chain using standard protocols (23). One 15-kb clone, LA26, was characterized by restriction enzyme mapping and hybridization with different cDNA inserts. Appropriate restriction fragments were subcloned into the pUC vector for further mapping and/or nucleotide sequencing. Exon-containing genomic subclones were sequenced in the pUC vector with the chain-termination method (24) using “universal” primers or sequence-specific oligonucleotides.

Polymerase Chain Reaction: Amplification and Sequencing of COL4A5 Exon 23”For sequencing analysis of the exon 23 region in genomic DNA from members of Alport kindred A21 and control samples, the exon-intron region was first amplified by PCR (26, 27). Briefly, 25 ng of DNA was used as template in each reaction. A pair of 27-mer primers was made from sequences starting 51 nucleotides and 55 nucleotides upstream and downstream from the exon, respec- tively. The primers were E23A 5’-AAGCTTACGTTATTGTGT-3’ (exon 23,5’ end) and E23B 5’-TGTAAAATGCCTTCCTTC-3’ (exon 23, 3’ end). The reactions were carried out using 1.25 unit of Ampli- Taq DNA polymerase (Perking-Elmer/Cetus) under conditions rec- ommended by the manufacturer.

For DNA sequencing, 1 p1 (-80 ng) of purified PCR product was added as template to the cycle-sequencing reaction mixture. The reaction mixture consisted of a d/ddNTP mix, 0.4-pmol primers E23A or E23B, labeled with fluoresceine (Fluore PrimeTM, Pharmacia LKB Biotechnology Inc.), 4 units of Ampli-Taq DNA polymerase (Perkin- Elmer/Cetus) in a cycle-sequencing buffer which contained 80 mM Tris-HC1, pH 8.9, at room temperature, 20 mM ammonium sulfate, and 5 mM MgClz. The reaction was carried out in microtuhes (2000 Micro Amp Reaction tubes, in a Perkin-Elmer/Cetus GeneAmp PCR system 9600). The total reaction volume was 5 pl. The conditions were 15 cycles of 95 “C for 15 s, 55 “C for 15 s, and 70 ”C for 45 s. This was followed by another 15 cycles of 95 “C for 15 s, and 70 “C for 45 s. The reaction mixtures were denatured with 4 r l of formamide at 95 “C for 3 min and sequenced on an automated laser fluoroscent DNA sequencer (Pharmacia).

Allele-specific Hybridization-Allele-specific hybridization was car- ried out to analyze the presence or absence of the mutation among the family members and controls. PCR-amplification of exon 23 was first performed, and duplicate filters were made by loading 5 pl of the PCR-amplified product to the two parallel slots of a slot-blot appa- ratus (Schleicher & Schuell, S&S Minifold 11) containing a nitrocel- lulose filter. Each filter was hybridized either with the ~ - ~ ~ P - l a b e l e d 15-mer normal (5’-ACAAGCTGGTGCAAC-3’) or mutant (5’-ACA- AGCTTGTGCAAC-3’) oligonucleotide probe a t 37 “C for 4 h. Wash-

I I I I 0 1 2 3

ing was performed once by 2 X SSPE, 0.1% sodium dodecyl sulfate a t room temperature and then soaking in the same solution a t 37 “C for 60 min for the mutant oligonucleotide or 90 min a t 37 “C for the normal oligonucleotide.

RESULTS

Full-length cDNA Clones for Collagen a5(IV)-Screening of the HT-1080 cell cDNA library resulted in several overlapping clones, one of which (HT-14) reached 2,499 bp upstream from our previously reported (15) PL-31 cDNA clone (Fig. 2) but only 9 bp upstream of clones reported by Pihlajaniemi et al. (16) which did not contain the complete 5’-end translated sequence. However, screening of the primer-extended human kidney cDNA library yielded one 0.72-kb clone, JZ-4, which contained 443 bp reaching further upstream from HT-14 into a 5’-end-untranslated region. The clones isolated here, to- gether with the previously reported ones, covered a total of about 6,5 kb, which is about the size of the mRNA as estimated by Northern hybridization (15).

Nucleotide and Predicted Amino Acid Sequence of the Hu- man a5(IV) Chain cDNA Clones-The 443-nucleotide-long new sequence of the presently isolated clones together with those of previously reported clones (15, 16) spans a total of 6,453 bp with a 203-bp 5”untranslated region, a 5,055-bp open reading frame starting with the ATG codon for methi- onine, and ending with a 1195-bp 3”untranslated sequence starting with a translation stop codon TAA and ending with an AIo tail (Fig. 3). The sequences provide the first complete amino acid sequence for the a5(IV) collagen chain from any species. There is a putative leucine-rich hydrophobic signal peptide of 26 residues. The signal peptide cleavage site was predicted using the computer program SIGSEQ (The Rocke- feller University) based on the method of von Hejne (28). The highest probability for the cleavage site was obtained between residues alanine 26 and alanine 27. The actual collagenous domain of the a5(IV) chain contains a total of 1430 amino acid residues and starts with a 14-residue noncollagenous sequence which contains 4 cysteine residues as the al(1V) and a2(IV) chains (6-8). Additionally, the Gly-Xaa-Yaa re- peat containing collagenous domain is interrupted at 22 lo- cations by 1-13-residue-long noncollagenous sequences as previously reported (15, 16). The carboxyl-terminal globular

I I I 4 5 6 kb

H T - 1 1 E l

J Z - 4 P L - 3 1

MD-6 E n r - 1 4 - 1 E n r . 1 4 . z E ~ ~ . i d - 3 1

I I I I

H T - 1 4

PL-35

5’ , I 1 3‘ ATG cDNA T A A A A A A A A P

1 2 7 1 4 5 7 1 6 8 5

r Collagenous domain NC-domain Signal peptide

FIG. 2. Illustration of overlapping cDNA clones spanning the entire mRNA for the human a5(IV) collagen chain and scheme of the polypeptide chain. Top, scale in kb. Middle, scheme of overlapping cDNA clones and the full-length cDNA. The cDNA clones PL- 31, MD-6, and PL-35 have been described previously (15). The HT-11 and HT-14 coding sequence contains three internal EcoRI restriction sites (E). The three 5’-end EcoRI restriction fragments of HT-14 are termed HT-14-1, HT-14-2, and HT-14-3. The translation initiation (ATG) and stop (TAA) codons are indicated as well as the poly(A) tail. Bottom, illustration of the primary structure of the human a5(IV) collagen chain. The signal peptide (residues 1-26) is indicated by a black pattern, the collagenous domain (residues 27-1456) by a white pattern interrupted by short noncollagenous sequences (shaded boxes), and the NC domain (residues 1457-1685) by a cross-hatchedpattern.

12478 Human a5(IV) Collagen Chain 1 AGGGGGGMGGMGAGTAGCTCCTTCTTCTTCTTCTTTTTTTTTTCTTC~CTCTT~GCTTCTTTCTCTTCACCCMG 82

8 3 CCTCACTGTCCCTCTCCGGCTCTAGCTCTCTCCATATAI\ACCCTCMGATTAIGTCMTTGGTTAGAGCCAGCCGGGMTTTCGTGCGGGTGCTGMGGAGCTGCGGGAGCCGGAGMG~ 203

2 0 4 ATGAAACTGCGTGGAGTCRCCTGGCTGCCGGCTTGTTCTTACTGGCCCTGAGTCTTTGGGGGCAGCCTGCAGAGGCTG I M K L R G V S L A A G L F L L A L S L W G Q P A E A A

324 ~ ~ G G G G I G G G A G A G A G A G G G T T T C C A G G T T T G G M G G A C A C C C A G G A T T G C C T G G A T T T C C A G G T C C A G M G r ~ C C T C C G G G G C C T C G G G G A C A A A A G G G T G A T G A T 4 4 3 I I S G I K G E K G R R G F P G L E G H P G L P G F P G P E G P P G P R G Q K G D D EO

4 1 4 GGARTTCCAGGGCCACCAU;ACC~~MTCAGAGGTCCTCCTGGACTTCCTGGATTTCCAGGGACACCAGGTCTTCCTGGMTGCCAGGCCACGATGGGGCCCCAGGACCTC~GGT 5 6 3 B I G I P G P P G P K G I R G P P G L P G F P G T P G L P G M P G H D G A P G P Q G 120

V

564 ATTCCCGGA GCMTGGMCCMGGGAGMCG~;GATTTCCAGGCAGTCCCGGTTTTCCTGGTTTACAGGGTCCTCCAGGACCCCCTGGGATCCCAGGTATG~G~TGMCCAGGTAGT 683 1 Z l I P G & N G T K G E R G F P G S P G F P G L O G P P G P P G l P G M K G E P G S 1 6 0

G P K G N P G Y P G P P G I Q G L P G P T G I P G P I G P P G P P 200 GGACCAAAGGGTIII\TCCAGGATATCCAGGTCCTCCTGGMTAC~GGCCTACCTGG~CCCACTGGTATACCAGGGCCRATTGGTCCCCCAGGACCACCA 803

Z O I G L M G P P G P P G L P G P K G N U G L N F G P K G E K G E Q G L Q G P P G P 2 4 0 804 GGTTTGATGGGCCCTCCTGGTCCACCAGGACTTCCAGGACCTMGGGGRRTRTGGGCTT T C C A A A G G T G A R R A A G G T G A G C I U L G G T C T T C A G G G C C C A C C T G G G C C A 9 2 3

924 CCTGGGCAGAT GGAGATCAGGGACTTCCTGGTGACCGAGGGCCTCCTGGACCTCCAGGGATACGTGGTCCTCCAGGTCCC 1043 G D Q G L P G D R G P P G P P G I R G P P G P 280

I

I 1

1044 C C A G G T G G T G A ~ T G A G R A G G G T G A G C ~ G G A G A G C C A G G C I G A G G T ~ C C A G G C ~ G A T G G A G ~ T G G C C ~ C C A G G ~ T T C C T G G T T T G C C T G G T G A T C C T G G T T A C 1 1 1 1163

2 8 1 P G G E K G E K G E Q G E P G K R G K P G K D G E N G Q P G I P G L P G D P G Y 320

1 1 6 4 CCTGGTGMCCCGGMGGGATGGTGAARI\GGGCC~GGTGACACTGGCCCACCTGGACCTCCTGGACTTGT 3 2 1 P G E P G R D G E K G Q K G D T G P P G P P G L V

I V

V 1 2 8 4 ATTGGGTTGCCTGGGTTGCCTGGAGAARRRGGAGAGCGAGGATTTCCTGGMTACAGGGTCCACCTGGCCTTCCTGGACCTCCAGGGGCTGC~GGTCCTCCTGGCCCTCCTGGA 1 4 0 3 361 I G L P G L P G E K G E R G F P G I Q G P P G L P G P P G A A V M G P P G P P G 4 0 0

V I

1404 TTTCCTGGAGAAAGGGGTCAGRAAGGTGATGARGGRCCACCTGG~TTTC~GACCTCCTGGACTTGACGGACAGCCTGGGGCTCCTGGGCTTCCAGGGCCTCCTGGCCCTGCT 1523 GA

4 0 1 F P G E R G Q K G D E G P P G I S I P G P P G L D G Q P G A P G L P G t ' P G P A 440 V I 1 c

1 5 2 4 GGCCCTCA TCCTrCTAGTGRTGA GGCCCTCCAGGCCCCCCAGGATCTCCAGGTGATRAAGGACTCCMGGAGAAC~GGAGTGIL4RGGTGAC~AGGTGACACT 1 6 4 3 A A

~ ~ ~ G P H I P P S D E G P P G P ~ G S P G D K G L Q G E O G V ~ G D R G D T 4 8 0 S

1 5 4 4 TGCTTCMCTGCATTGGRACTGGTATTTCRGGGGCCTCCAGGTCMCCTGGTTTGCCAGGTCTCCCAGGTCCTCCAGGATCTCTTGGTTTCCCTGGACAGRAAGGGG~GGA~~G~T 1 7 6 3 4 8 l [ C ) F N ( C ) I G T I G I S G P P G Q P G L P G L P G P P G S L G F P G Q K G E K G Q 7 5 2 0

V I 1 1

I X 1 1 6 4 ~ G T G C " C T G G T C C C R A A G G A T T A C C A G G C A T T C C A G G A G C T C C A G G T G C T C C ~ T C C T G G A T C T A A A G G T G M C C T G G T G A T A T ~ T T T ~ C ~ M T G M G G G T G A C R R R 1 8 8 3

5 2 1 G A T G P K G L P G l P G A P G A P G F P G M K G @ K 5 6 0 X

1 8 8 4 GGAGAGTTGGGTTCCCCTGGAGC'TCCRGGGCTTCCTGGTTTACCTGGCACTCCTGGACAGGATGGATTGCCAGGGCTTCCTGGCCCG~GGAGAGCCTGGTGGMT 5 6 1 G E L G S P G A P G L P G G P P G P G Q D G L P G L P G P K G E P G G 1 ~ ~ ~ ~ T 2 ~ ~ ~

2 0 0 4 GRAAGAGGTCCCCCTGGGMCCCAGGTTTACCAGGCCTCCCAGGGMTATAGGGCCTATGGGTCCCCC GGTT CGGCCCTCCAGGCCCAGTAGGTGAAAAAGGCATACAGGTGTGGCA 2 1 2 3 6 0 1 E R G P P G N P G l . P O L P G l ~ I G P M G P P ~ G ~ C ~ ~ G P V G E K G 1 Q G V A L A L Q 64C

X I

XI1

2124 U;MTCCAGGCCAGCCAGGRATRCCAGGTCCTIL4RGGGGATCCAGGTCAGACTAT~CCCAGCCGGGG~GGCTTGCCTGGTMCCCAGGCAGAGATGGTGATGTAGGTCTTCCA 2 2 4 3 5 4 1 G N P G Q P G I P G P K G D P G Q T l I T Q P ) G K P G L P G N P G R D G D V G L P 680

C G

X l l l F R

2 4 8 4 CCACCAGGACTTCCAGGTTTClVULGGAGCACTTGGTCClVLRRGGTGATCGTGGTTTCCCAGGACCTCCGGGTCCTCCAGGACGCACTGGCTTAGATGGGCTCCCTGGACC~GGTGAT 2 6 0 3 7 6 1 P P G L P G F K G A L G P K G D R G F P G P P G P P G R T G L D G L P G P K G D 800

.. .

2604 G T T G G A C C R R R T G G A C ~ C C T G G A C C I V \ T G G G A C C T C C T G G G C T G C C ~ G A C C A C C A G G A C C A C C A G G G A T T C C T G G G C C R A T A G G T C I \ I \ C C T G G T T T A C A T G G A 2 1 2 3 E O l V G P N G Q P G P M G P P G L P G I G V Q G P Q G P P G I P G P I G Q P G L H G 8 4 0

2 1 2 4 ATACCAGGAGAGMGGGGGATCCAGGACCTCCTGGACTTGATGTTCCAGGACCCCCAGGTGARAGAGGCAGTCCAGGGATCCCCGGAGCACCTGGTCCTATAGGACCTCCAGGATCAC~A 2 8 4 3 8 4 1 1 P G E K G D P G P P G L D ~ G P P G E R G S P G l P G A P G P l G P P G S P 880

X V I

CGG X V l l

2 8 4 4 GGGCTTCCAGGRAAAGCAGGTGCCTCTGGATTTCCAGGTACCIGGTGRRRTGGGTATGATGGGACCTCCAGGCCCACCAGGACCTTTGGG~TTCCTGGCAGGAGTGGTGTACCTGGT 2 9 6 3 8 8 1 G L P G K A G A S G F P G T K G E M G M M G P P G P P G P L G I P G R S G V P G 9 2 0

R

2 9 6 4 CTTAAAGGTGATGATGGCTTGCAGGGTCAGCCAGGACTTCCTGGCCCTACAGGAGRGGTAGT~GGAGAGCCTGGCCTTCCAGGCCCTCCTGGACCM~GGATCC~TC 9 2 1 L K G D D G L Q G Q P G L P G P T G E K G S K G E P G L P G P P G P M ~ ~ ~ ~

3084 GGCTCAAAAGGAGAGAAGGGGG X V l l l

9 6 1 G S K G E K G E P G L P G I P G V S G P K G Y Q G L P G D P G Q P G L S G Q P G

1 0 0 1 L P G P P G P K G M P G L P G Q P G L I G P P G L K G T I G D M G F P G P Q G V

1 0 4 1 E G P P G Q S G C . P G Q P G S P G L P G Q K G D K G D Q G I S ~ G L P G L P G

1 0 8 1 P K G E P G L P G Y P G N P G I K G S V G D P G A K G o P G L X I X

~ I ~ ~ P G F P G T F G F P G Q K G I S G P P G N P G L P G E P G P V G G G G ~ P G Q P

L l h l G P P G E K G K P C O U G I P G P A C O K G E P G Q P G F G N P G P P G L P G L

~ ~ O ~ S G Q K G D G G L P G I P G N P G L P G P K G E P G F H ~ ~ G V Q G P ~ G ~ P

I ~ ~ I G S P G P A ~ G P K G N P G P Q G P P G R P G L P G P E G ~ ~ G L ~ G N G G ~ xx

~ Z E ~ K G E K G N P G Q P G L P G L P G L K G D Q G P P G L Q G N ~ G R ~ G L N G ~ K X X I

I ~ ~ I G D P G L P G V P G F P G M K G P S G V P G S A G P E G E ~ G L ~ G ~ ~ G ~ ~ G

~ ~ ~ ~ L P G P S G Q S I I I I K ] G D A G P P G I P G Q P G L K G L ~ G ~ Q G ~ Q G ~ , ~ G

~ ~ O ~ P T G P P G D P G R N G L P G F D G A G G R K G D P G L P G Q ~ G T R G L D ~ ~ X X l l

3083 9 6 0

3203 1000

1040

1080

1120

1 1 6 0

1200

1240

1280

1 3 2 0

1360

1 4 0 0

1 4 4 0

1 4 4 1 P G P D G L Q G P P G P P G T S I S V A H G F L I T R H S Q T T D A P Q @ P Q G T

1520 1 4 8 1 L Q V Y E G F S L L Y V Q G N K R A H G Q D L G T A G S @ L R R F S T M P F M F

1480

1 6 0 1 0 U K R T 1685

FIG. 3. Complete amino acid sequence of the human a5(IV) collagen chain. Upper line, nucleotide sequence (nucleotides 1-3105) of the cDNA clones characterized in this study to the 3' end of the HT-14 cDNA clone which overlaps by 163 nucleotides the previously

Human aS(IV) Collagen Chain 12479

noncollagenous domain (NC domain) contains 229 residues as reported earlier (15,16). The complete a5(1V) chain trans- lation product contains 1685 residues, and the actual mature chain contains 1659 residues. The calculated molecular weight of the mature human a5(IV) chain is 158,303.

The sequence of HT-14 determined here shows several differences from the corresponding sequence published else- where (16) and as indicated in Fig. 3. First, the codon GCC (nucleotides 546-548) is for alanine (residue 115), not valine. Second, nucleotides 2078-2087 differ, changing amino acid residues 625-628 from Leu-Ala-Leu-Gln to Phe-Gly-Pro-Pro. Accordingly, interruption XI1 is relocated and contains amino acids Gly-Phe instead of Leu-Gln. Third, nucleotides 2,204 (C) and 2,206 (G) were found to be G and C, respectively, changing amino acid residues 667 and 668, respectively, from Phe + Leu and Arg + Pro. Finally, nucleotides 2,465-2,467 were changed from CGG to GCC converting residue 888 Arg to Ala.

HindIII RFLP in the Sequence Encoded by cDNA Clone HT-14-2 in an Alport Patient-As a part of a study to identify and characterize mutations in the COLAA5 gene in patients with Alport syndrome, genomic DNA from members of kindred A21 was digested with HindIII, electrophoresed, Southern blotted, and hybridized with a mixture of 32P-labeled cDNA inserts (JZ-4, HT-14, PL-31, MD-6, PL-35) spanning the entire a5(JV) chain coding sequence. In the affected male (IV,) from the Danish kindred A21, there was a loss of a 1.35- kb fragment and appearance of a variant 1.25-kb band not seen in controls (Fig. 1). Repeated hybridization analyses with individual cDNA inserts demonstrated that the poly- morphism was present in the region of the COLAA5 gene coding for sequences contained in HT-14-2. Furthermore, analysis of DNA from several members of kindred A21 dem- onstrated that the proband’s female cousin (IVl) was hetero- zygous having the normal 1.35-kb as well as the variant 1.25- kb band, whereas her two sisters (IV2 and IV3) and the daughter of IVl (VI) only had the 1.35-kb band, and, therefore, is homozygous for the normal allele. Normally, HindIII di- gested genomic DNA reveals four constant bands of approxi- mately 6.2, 5.1,3.8, and 1.35 kb after hybridization with HT- 14-2 (Fig. 1).

Sequencing of Exon 23 Region Encoded by a Part of HT-14- 2 and Identification of a Point Mutation in Alport Kindred A21”It was considered a possibility that the variant 1.25-kb HindIII restriction fragment observed in the proband was caused by a point mutation creating a novel HindIII site in the sequence coding for HT-14-2. We, therefore, searched the HT-14-2 sequence (nucleotides 1136-2360, Fig. 3) for se- quences resembling the HindIII recognition sequence AAGCTT. Five such sequences differing by only one base could be found (underlined in Fig. 3). Furthermore, based on the RFLP data, the putative mutated sequence should be in an exon contained in a 1.35-kb genomic HindIII fragment. Characterization of genomic clone LA26 containing a part of the COLZAS gene revealed that it contained, indeed, a 1.35- kb HindIII fragment (not shown) which was subcloned and

sequenced from both ends. The sequencing demonstrated that this fragment contained one 71-bp exon (nucleotides 1720- 1790, Figs. 3 and 4) which contained one of the five sequences differing by one base from the HindIII recognition sequence (AAGCTG, Fig. 4). This exon was shown to be the 23rd as counted from the 5’ end of the gene, based on further analysis of the gene (30); Exon 23 starts with the second base for the codon of glycine 506 and ends with the third base of the codon for proline 529. It is, therefore, similar to a corresponding exon in the COIAAl gene (31).

Having determined the nucleotide sequence of the 1.35-kb HindIII restriction fragment containing exon 23 and 51 bases upstream to the HindIII site and 55 bases downstream, this exon region of the COLAA5 gene from the proband of kindred A21 could be subjected directly to DNA sequencing following PCR amplification, since he is hemizygous for this allele. The sequencing results revealed a single base (base 1764) change in the coding strand of exon 23 with a substitution of G + T (Fig. 4). The finding was confirmed by repeated sequencing from both strands, The mutation changes the GGT codon of glycine 521 to a codon TGT for cysteine. Furthermore, the mutation created a new HindIII restriction enzyme cleavage site as hypothesized. DNA sequencing of PCR-amplified exon 23 region from the heterozygote female cousin indicated the presence of both the normal and mutated alleles (data not shown). The results suggested that the HindIII restriction site observed in the proband and his female cousin with cDNA insert HT-14-2 was indeed due to the mutation in exon 23. Accordingly, the mutation should be present in one allele of the cousin but not the other family members available for the study or other unaffected individuals. In order to confirm this, we applied allele-specific hybridization of PCR products with a normal 15-mer oligonucleotide and 15-mer oligonucleotide containing the G -P T mutation in the center. Results from the slot blot analyses are shown in Fig. 5. It can be seen that DNA from the proband hybridized strongly, and that of his female cousin hybridized quite strongly with the mutation specific probe, whereas DNA from other family members and one unaffected control did not at all. In contrast, the pro- band’s DNA did not hybridize with the normal probe, the cousin hybridized quite strongly, and other unaffected indi- viduals hybridized very strongly.

DISCUSSION

Human a5(IV) Collagen Chain-The present work provides the complete primary structure for the human a5(IV) collagen chain whose gene has been shown to be mutated in several patients with X chromosome-linked Alport syndrome. Com- parison of some of the characteristics of the a5(IV) chain with those of the previously sequenced human and murine al(IV) and a2(IV) chains (6-8, 32-35) and the partially characterized bovine a3(IV) chain (11) is summarized in Table I. The complete a5(IV) chain translation product con- tains 1,685 residues but 16 less residues for the al(1V) chain

J. Zhou, A. Leinonen, L. T. Chow, and K. Tryggvason, unpub- lished results.

~

reported sequence of PL-31 (15). Lower line, entire amino acid sequence of the a5(IV) chain deduced from the nucleotide sequence of the presently and previously (15, 16) isolated clones providing the accurate residue numbers starting at the translation initiator methionine. Nucleotides reported in Ref. 16 differing from the sequence of the cDNA clones isolated here are shown above the nucleotide sequence and amino acid residues reported in Ref. 16 differing from the sequence determined here are indicated below the sequence. The numbering of nucleotide sequences starts from at the first nucleotide of JZ-4. The putative cleavage site of signal peptidase is indicated by an arrow. The cysteine residues are circled and interruptions in the Gly-Xaa-Yaa repeat sequence are boxed and numbered from the amino-terminal end by Roman numerals. The 14-residue noncollagenous sequence at the amino terminus of the mature chain is boxed as is the carboxyl-terminal NC domain. Nucleotide sequences within the cDNA fragment HT-14-2 which differ from the HindIII cleavage site by one base (see text) are underlined.

~~~ ~

12480 Human a5(IV) Collagen C h i n 1,7,1 9

T T C C C ? G G A C W ~ G G G G , M I V \ i \ F G A C " ? 6 G 1 G g ~ a P h e ? ~ ~ G l y G 1 1 L y ~ S l y G ~ ~ ~ y ~ ~ l y ~ : ~ . ; ~ ~ . ~ ~ : y A l ~ ? ~ ~ G l y ~ : ~ ~ ~ ~ ~ l y L ~ ~ ? : ~

5 2 9 ..,. I . . _ I

5 2 1 B t a B ( i : r Z : C d g Z a C B ~ i d l a a a a c d a a a d g a a a 2 a ( i ~ ~ : ~ ~ ~ ~ -

E23B

FIG. 4. Nucleotide sequence of exon 23 (capital letters) of the gene and its deduced amino acid sequence and mutation observed in Alport kindred A21. About 50 nucleotides (lower- case letters) from the adjacent introns are shown. The numbering of coding nucleotides and amino acid residues according to Fig. 3. The intron sequences used for design of PCR primers E23A and E23B are indicated by arrows. A HindIII cleavage site upstream of the exon is underlined as is the sequence within the exon which is changed to a HindIII site by the mutation. The mutation at nucleotide 1763 converts glycine 521 to cysteine.

Control Mutant

Normal male

Proband male [ I V - 4

cousin I V - 1

Female cousin I V - 2

cousin I V - 3

Cousin's daugther f v - 1 [ -

FIG. 5. Allele-specific hybridization of PCR-amplified exon 23. DNA from the proband (hemizygote) and his female cousin (heterozygote) are positive with the mutant oligonucleotide probe, and the cousin's DNA is also positive with the normal probe. Other family members and a normal male control are positive only with the normal control probe.

TABLE I Comparison of mammalian type ZV collagen Q chains

a5" alh a2' a3d 014'

Complete translation product 1685 1669 1712

Mature Q chain (residues) 1659 1642 1676 Signal peptide (residues) 26 27 36 Collagenous domain (residues) 1430 1413 1449 NC domain (residues) 229 229 227 233 231 Number of interruptions in

(residues)

collagenous domain 22 21 23

a Refs. 15 and 16 and this study. Refs. 6 and I. Ref. 8. Ref. 11. ' Ref. 14.

and 27 more residues for the a2(IV) chain. The processed mature chains have 1659, 1642, and 1676 residues, respec- tively. The calculated molecular weight of the processed

a5(IV) chain is 158,303 and that of the al(1V) and a2(IV) chains is 157,625 (6) and 163,744 (8), respectively. The a5(IV) and al(1V) chains which have been shown to be more closely related with each other than to the other type IV collagen a chains (15, 16) have signal peptides of identical size and NC domains. Initial characterization of the 3' end of the human COLAAS gene (30) has previously also demonstrated this close evolutionary relationship by showing that the COLAA5 and COL4Al genes have practically identical exon size patterns in this part of the gene. In contrast, the COL4A2 gene has diverged quite extensively from the other two. The sequence of exon 23 of the COUA5 gene determined in this study is identical in size to the corresponding 71-bp exon 24 in the COL4Al gene (31), thus further emphasizing the evolutionary relationship of the COL4A5 and COL4Al genes.

To date, it is not known whether the a5(IV) collagen chain is present in homotrimer triple-helical molecules or whether it is incorporated into heterotrimers together with other type IV collagen a chains. Due to the low abundance of the a5(IV) chain in tissues and also because of the insolubility of base- ment membranes in general, proper protein chemical analyses on the molecular structure are difficult to carry out. The availability of full-length cDNA clones enables expression of the a5(IV) collagen chain as well as the other collagen IV a chains in eukaryotic systems for such studies.

Mutations in the COMA5 Gene in Alport Syndrome-In the present study we identified a novel point mutation in the COLAA5 gene in Danish kindred A21, converting glycine 521 in the collagenous domain of the a5(IV) chain to cysteine. Although no protein data are available, it is likely that the mutation is causative for the disease in this kindred for several reasons. First, the mutation was only found in the proband and his heterozygous female cousin who has both proteinuria and hematuria. Second, the substitution of glycine for cysteine can be anticipated to have several severe effects on a type IV collagen molecule containing such a mutated chain. For ex- ample, a glycine substitution can markedly disturb the folding of the triple helix, and the introduction of a cysteine residue into the chain can lead to abnormal intra- or intermolecular disulfide bonds that interfere with extracellular supramole- cular assembly of type IV collagen. This is supported by the identification of similar glycine mutations in type I collagen in patients with Osteogenesis imperfectu. To date, there are at least 20 examples of mutations resulting in glycine + cysteine substitutions in the al(1) and a2(I) chains of type I collagen in 0. imperfectu (see Ref. 36). These mutations which have been located to different regions of the a chain can cause mild to lethal forms of the disease. Although a certain mutation in the COL4A5 gene can be considered with high certainty to cause malfunction of the triple-helical type IV molecule, it is still not well understood how the different mutations can lead to quite different phenotypes. Including the mutation char- acterized here, five mutations, both deletions and point mu- tations, have thus far been found in the COLAAS gene in patients with Alport syndrome and characterized in detail. These mutations are illustrated in Fig. 6 with the correct a5(IV) chain residue numbering elucidated in this study. In one patient with hematuria and severe hearing loss a deletion of exons 5 to 10 from the 3' end resulted in the loss of 240 residues (i.e. residues 1263-1502) from both the collagenous and NC domains (2). In another kindred, replacement of cysteine 1564 by serine, possibly leading to weakening of intermolecular cross-links, leads to adult onset end stage renal disease (ESRD) with hearing loss (2, 3). The substitution of serine for tryptophan 1538, conserved in all known type IV collagen a chains from Drosophila to man, leads to early type

Human a5fIV) Collagen Chain 12481 A r g Cys

t t A s p t t

Ser

G 'y325 G1Y521 G1y1143 cys1564

1

TrP1538 t

S e r

1 5

1 2 6 3 1 5 0 3

FIG. 6. Illustration of known mutations in the human a5(IV) collagen chain in different Alport patients. Top, amino acid substitutions resulting from five different point mutations. Bottom, consequence of the deletion of exons 5-10 (from the 3' end of the gene) resulting in the loss of 240 amino acid residues. For references and phenotypes, see "Discussion."

onset ESRD with no hearing loss but GBM splitting (37). The replacement of glycine 1143 by aspartate leads to an atypical form of Alport syndrome with juvenile onset and GBM changes but absence of hearing loss or ocular lesions (21). And finally, a single-base mutation caused the conver- sion of glycine 325 to arginine in a kindred with juvenile onset ESRD, hearing and GBM splitting, but without ocular le- s i o n ~ . ~ The mutation characterized here converting glycine 521 to cysteine causes a disease in the hemizygote with juvenile onset, GBM changes, and hearing loss but no ocular lesions.

It is likely that accumulating information on novel muta- tions in the COMA5 gene and their resulting phenotypes will shed some light on the mechanisms of phenotype generation. However, analysis on the properties of type IV collagen mol- ecules containing a5(IV) chains as well as their mutated forms are essential for elucidating the molecular pathology of such molecules. The generation of full-length cDNA clones, their expression in eukaryotic systems, assembly of type IV collagen isoforms, and studies of their properties with and without mutations may enable such in uitro analysis. Furthermore, the use of transgenic mice with mutation in the COMA5 gene made by gene targeting will be useful for this purpose.

Acknowledgments-We thank Anette Thomsen for excellent tech- nical assistance and the Department of Nephrology, Odense Sygehus, for referral of the proband.

Knebelmann. B.. Deschenes. G.. Gros. F.. Hors. M.-C. . Griinfeld.

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