Hum Genet (1992) 90: 467-468
human .. geneucs 9 Springer-Verlag 1992
a-l-Antichymotrypsin variant detected by PCR-single strand conformation polymorphism(PCR-SSCP) and direct sequencing Michio Tsuda 1, Yukari Sei 1, Masahiko Matsumoto 1, Hiroshi Kamiguchi 2, Masahiro Yamamoto 3, Yukito Shinohara 3, Tsuyoshi Igarashi 4, Masaichi Yamamura 1 1Department of Molecular Life Science, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 2Biochemistry Laboratory, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 3Department of Neurology, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 4Osaka Teishin Hospital, Tennnouji, Osaka, 543 Japan Received: 6 May 1992 / Revised: 22 July 1992
Abstract. A new m u t a n t a - l - a n t i c h y m o t r y p s i n (variant A C T ) was f o u n d by p o l y m e r a s e chain reaction single strand c o n f o r m a t i o n p o l y m o r p h i s m and direct sequencing. In this variant A C T , two bases ( A A ) were deleted f r o m c o d o n 391. This resulted in a different a m i n o acid sequence d o w n s t r e a m of the deletion point, elongating the peptide chain by 10 amino acids.
Introduction ~ - l - A n t i c h y m o t r y p s i n ( A C T ) is a plasma protease inhibitor belonging to the serpine (serine protease inhibitor) superfamily. It is present in n o r m a l h u m a n serum at high concentrations (294 +_ 44 gg/ml). T h e physiological activity of A C T is not yet k n o w n , although it has b e e n reported that A C T has m a n y functions in vitro (Hudig et al. 1981; M a t s u m o t o et al. 1981; T a k a d a et al. 1988; T s u d a et al. 1982, 1986; Travis et al. 1978). T h e r e f o r e , qualitative and quantitative changes in A C T are considered to cause some dysfunction of the physiological response, resulting in specific diseases. Recently, we described a variant A C T frequently f o u n d in patients with occlusivecerebrovascular disease (Tsuda et al. 1992). H e r e , we report a n o t h e r m u t a n t A C T , caused by the deletion of two bases ( A A ) .
were 94~ for 2 min to denature the DNA, then 30 cycles at 94~ for 1 min, 55~ for 1 min, 72~ for 2 rain, and a single cycle at 72~ for 5 min. The reaction mixture (100 gl) contained 100 pmol primers, dNTPs at 200gM, 1.5mMMgCI2, 0.5 ~g leucocyte genomic DNA. After 2% agarose electrophoresis, the PCR product was extracted, and the sequence was determined by the asymmetric PCR method. Thirty cycles of a second PCR were performed under the same conditions as the first, except that an unequal ratio of the two primers (100 : 1) was used. The second PCR product was purified on an ULTRAFREE (C3TK, Millipore) to remove the primers. The product was then sequenced with the AmpliTaq Sequencing Kit (TAKARA) or Sequenase DNA sequencing kit (version 2.0, USB) following the manufacturer's protocols.
PCR single strand conformation polymorphism analysis of exon V of the A CT gene PCR-single strand conformation polymorphism (SSCP) analysis was performed according to the method previously described (Tsuda et al. 1992). Primers labeled at the 5'-end with 32p were used for 30 PCR cycles. The PCR product was diluted 1 : 20 in the presence of 0.1% sodium dodecyl sulfate and 10mMEDTA. Then, 4 ~tl of this diluted solution was mixed with an equal volume of the denaturation solution (95% formamide, 20raM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), and heated to 90~ for 3 min. The denatured PCR product was applied to a 6% poly-
Materials and methods Polymerase chain reaction conditions and sequencing The polymerase chain reaction (PCR) was carried out using the following primers; 5'-TTACTGAGAGCCCCACTGCATGAT-3' and 5'-CATAAGGCTGTGCTTGATGTA-3'. PCR conditions
Correspondence to: M. Tsuda
Fig. 1. PCR-SSCP analysis of exon V of ACT gene. PCR products were denatured, applied to a 6% polyacrylamide gel containing 10% glycerol, and electrophoresed at room temperature. N normal; 1 ACT Isehara-1; 2 ACT Isehara-2
468 Normal
TTC ATG AGC AAA GTC ACC AAT CCC AAGCAAGCC TAG Phe Met Ser Lys Val Thr Asn Pro Lys Gln Ala *** 389 398
TTC ATG AGC I I 9 CAC CAA TCC CAA GCA AGC CTA GAG CTT GCC ATC AAG CAGTGG GGC TCT CAG TAA ACT lsehara-2 Phe Met Ser Ser His Gin Ser Gin Ala Ser Leu Glu Leu Ala Ile Lys Gin Trp Gly Ser Gin ~"** 389 391 408
Fig. 2. Amino acid sequence of ACT Isehara-2. The nucleotide and amino acid sequences of a part of exon V are displayed. 9 indicates the deleted bases. Asterisks indicate the terminal codon
Acknowledgements. This study was supported by the Osaka Foundation for Promotion of Clinical Immunology. We are grateful to Nadia El Borai for correcting the manuscript.
acrylamide gel containing 10% glycerol, and electrophoresed at room temperature.
References
Results and discussion T h e n u c l e o t i d e s e q u e n c e of h u m a n A C T has b e e n d e t e r m i n e d ( C h a n d r a et al. 1983: R u b i n et al. 1990). W e have r e p o r t e d o n e v a r i a n t A C T , A C T I s e h a r a - 1 , which was a p o i n t m u t a t i o n at c o d o n 389, resulting in a valine to m e t h i o n i n e t r a n s v e r s i o n ( T s u d a et al. 1992). P C R - S S C P analysis was u s e d to find o t h e r m u t a t i o n s of e x o n V containing the r e a c t i v e site o f the p r o t e a s e i n h i b i t o r . In 121 u n r e l a t e d individuals, this analysis s h o w e d o n l y o n e abn o r m a l p a t t e r n that was d i f f e r e n t f r o m native and A C T I s e h a r a - 1 D N A (Fig. 1). T h e s e q u e n c i n g o f the P C R p r o d u c t s with the new a b n o r m a l P C R - S S C P p a t t e r n rev e a l e d that it was h e t e r o z y g o u s in the A C T gene. This variant A C T g e n e h a d a d e l e t i o n of two bases ( A A ) at c o d o n 391, resulting in a d i f f e r e n t a m i n o acid s e q u e n c e d o w n s t r e a m o f the d e l e t i o n a n d 10 e x t r a a m i n o acids (408 a m i n o acids) c o m p a r e d with native A C T (398 a m i n o acids) (Fig. 2). T h e i n d i v i d u a l ( m a l e , 26 y e a r s old) with the n e w v a r i a n t A C T I s e h a r a - 2 has no a b n o r m a l s y m p t o m s at p r e s e n t . H o w e v e r , the c o n c e n t r a t i o n of A C T in his s e r u m was 110 ~tg/ml, a b o u t 40% of the n o r m a l level, suggesting that A C T I s e h a r a - 2 m i g h t n o t be s e c r e t e d f r o m the liver o r that it m a y b e r a p i d l y d e g r a d e d in the circulation.
Chandra T, Stackhouse R, Kidd VJ, Robson KJ, Woo SL (1983) Sequence homology between human c~l-antichymotrypsin, ulantitrypsin, and antithrombin III. Biochemistry 22:5055-5061 Hudig D, Haverty T, Fulcher C, Redelman D, Mendelson J (1981) Inhibition of human natural cytotoxicity by macromolecular antiproteases. J Immunol 126:1569-1574 Matsumoto M, Tsuda M, Kusumi T, Takada S, Shimamura T, Katsunuma T (1981) Enhancement by c~-l-antichymotrypsin of antibody response in vivo. Biochem Biophys Res Commun 100 : 478-482 Rubin H, Wang ZM, Nickbarg EB, McLarney S, Naidoo N, Shoenberger OL, Johnson JL, Cooperman BS (1990) Cloning, expression, purification and biological activity of recombinant native and variant human ~.l-antichymotrypsins. J Biol Chem 265:1199-1207 Takada S, Tsuda M, Yamamura M. Katsunuma T (1988) Effect of alpha-l-antichymotrypsin on activity of DNA primase isolated from human stomach adenocarcinoma cells. Biochem Int 16: 949-954 Travis J, Bowen J, Baugh R (1978) Human cq-antichymotrypsin: purification and properties. Biochemistry 17:5651-5656 Tsuda M, Ohkubo T, Kamiguchi H, Suzuki K, Nakasaki H, Mitomi T, Katsunuma T (1982) Purification, properties and identification of a serum DNA binding protein (64DP) and its microheterogeneity. Tokai J Exp Clin Med 7:201-211 Tsuda M, Masuyama M, Katsunuma T (1986) Inhibition of human DNA polymerase a by a~-antichymotrypsin. Cancer Res 46: 6139-6142 Tsuda M, Sei Y, Yamamura M, Yamamoto M, Shinohara Y (i992) Detection of a new mutant a-l-antichymotrypsin in patients with occlusive-cerebrovascular disease. FEBS LETT 304 : 66-68
human .. geneucs 9 Springer-Verlag 1992
a-l-Antichymotrypsin variant detected by PCR-single strand conformation polymorphism(PCR-SSCP) and direct sequencing Michio Tsuda 1, Yukari Sei 1, Masahiko Matsumoto 1, Hiroshi Kamiguchi 2, Masahiro Yamamoto 3, Yukito Shinohara 3, Tsuyoshi Igarashi 4, Masaichi Yamamura 1 1Department of Molecular Life Science, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 2Biochemistry Laboratory, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 3Department of Neurology, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11 Japan 4Osaka Teishin Hospital, Tennnouji, Osaka, 543 Japan Received: 6 May 1992 / Revised: 22 July 1992
Abstract. A new m u t a n t a - l - a n t i c h y m o t r y p s i n (variant A C T ) was f o u n d by p o l y m e r a s e chain reaction single strand c o n f o r m a t i o n p o l y m o r p h i s m and direct sequencing. In this variant A C T , two bases ( A A ) were deleted f r o m c o d o n 391. This resulted in a different a m i n o acid sequence d o w n s t r e a m of the deletion point, elongating the peptide chain by 10 amino acids.
Introduction ~ - l - A n t i c h y m o t r y p s i n ( A C T ) is a plasma protease inhibitor belonging to the serpine (serine protease inhibitor) superfamily. It is present in n o r m a l h u m a n serum at high concentrations (294 +_ 44 gg/ml). T h e physiological activity of A C T is not yet k n o w n , although it has b e e n reported that A C T has m a n y functions in vitro (Hudig et al. 1981; M a t s u m o t o et al. 1981; T a k a d a et al. 1988; T s u d a et al. 1982, 1986; Travis et al. 1978). T h e r e f o r e , qualitative and quantitative changes in A C T are considered to cause some dysfunction of the physiological response, resulting in specific diseases. Recently, we described a variant A C T frequently f o u n d in patients with occlusivecerebrovascular disease (Tsuda et al. 1992). H e r e , we report a n o t h e r m u t a n t A C T , caused by the deletion of two bases ( A A ) .
were 94~ for 2 min to denature the DNA, then 30 cycles at 94~ for 1 min, 55~ for 1 min, 72~ for 2 rain, and a single cycle at 72~ for 5 min. The reaction mixture (100 gl) contained 100 pmol primers, dNTPs at 200gM, 1.5mMMgCI2, 0.5 ~g leucocyte genomic DNA. After 2% agarose electrophoresis, the PCR product was extracted, and the sequence was determined by the asymmetric PCR method. Thirty cycles of a second PCR were performed under the same conditions as the first, except that an unequal ratio of the two primers (100 : 1) was used. The second PCR product was purified on an ULTRAFREE (C3TK, Millipore) to remove the primers. The product was then sequenced with the AmpliTaq Sequencing Kit (TAKARA) or Sequenase DNA sequencing kit (version 2.0, USB) following the manufacturer's protocols.
PCR single strand conformation polymorphism analysis of exon V of the A CT gene PCR-single strand conformation polymorphism (SSCP) analysis was performed according to the method previously described (Tsuda et al. 1992). Primers labeled at the 5'-end with 32p were used for 30 PCR cycles. The PCR product was diluted 1 : 20 in the presence of 0.1% sodium dodecyl sulfate and 10mMEDTA. Then, 4 ~tl of this diluted solution was mixed with an equal volume of the denaturation solution (95% formamide, 20raM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), and heated to 90~ for 3 min. The denatured PCR product was applied to a 6% poly-
Materials and methods Polymerase chain reaction conditions and sequencing The polymerase chain reaction (PCR) was carried out using the following primers; 5'-TTACTGAGAGCCCCACTGCATGAT-3' and 5'-CATAAGGCTGTGCTTGATGTA-3'. PCR conditions
Correspondence to: M. Tsuda
Fig. 1. PCR-SSCP analysis of exon V of ACT gene. PCR products were denatured, applied to a 6% polyacrylamide gel containing 10% glycerol, and electrophoresed at room temperature. N normal; 1 ACT Isehara-1; 2 ACT Isehara-2
468 Normal
TTC ATG AGC AAA GTC ACC AAT CCC AAGCAAGCC TAG Phe Met Ser Lys Val Thr Asn Pro Lys Gln Ala *** 389 398
TTC ATG AGC I I 9 CAC CAA TCC CAA GCA AGC CTA GAG CTT GCC ATC AAG CAGTGG GGC TCT CAG TAA ACT lsehara-2 Phe Met Ser Ser His Gin Ser Gin Ala Ser Leu Glu Leu Ala Ile Lys Gin Trp Gly Ser Gin ~"** 389 391 408
Fig. 2. Amino acid sequence of ACT Isehara-2. The nucleotide and amino acid sequences of a part of exon V are displayed. 9 indicates the deleted bases. Asterisks indicate the terminal codon
Acknowledgements. This study was supported by the Osaka Foundation for Promotion of Clinical Immunology. We are grateful to Nadia El Borai for correcting the manuscript.
acrylamide gel containing 10% glycerol, and electrophoresed at room temperature.
References
Results and discussion T h e n u c l e o t i d e s e q u e n c e of h u m a n A C T has b e e n d e t e r m i n e d ( C h a n d r a et al. 1983: R u b i n et al. 1990). W e have r e p o r t e d o n e v a r i a n t A C T , A C T I s e h a r a - 1 , which was a p o i n t m u t a t i o n at c o d o n 389, resulting in a valine to m e t h i o n i n e t r a n s v e r s i o n ( T s u d a et al. 1992). P C R - S S C P analysis was u s e d to find o t h e r m u t a t i o n s of e x o n V containing the r e a c t i v e site o f the p r o t e a s e i n h i b i t o r . In 121 u n r e l a t e d individuals, this analysis s h o w e d o n l y o n e abn o r m a l p a t t e r n that was d i f f e r e n t f r o m native and A C T I s e h a r a - 1 D N A (Fig. 1). T h e s e q u e n c i n g o f the P C R p r o d u c t s with the new a b n o r m a l P C R - S S C P p a t t e r n rev e a l e d that it was h e t e r o z y g o u s in the A C T gene. This variant A C T g e n e h a d a d e l e t i o n of two bases ( A A ) at c o d o n 391, resulting in a d i f f e r e n t a m i n o acid s e q u e n c e d o w n s t r e a m o f the d e l e t i o n a n d 10 e x t r a a m i n o acids (408 a m i n o acids) c o m p a r e d with native A C T (398 a m i n o acids) (Fig. 2). T h e i n d i v i d u a l ( m a l e , 26 y e a r s old) with the n e w v a r i a n t A C T I s e h a r a - 2 has no a b n o r m a l s y m p t o m s at p r e s e n t . H o w e v e r , the c o n c e n t r a t i o n of A C T in his s e r u m was 110 ~tg/ml, a b o u t 40% of the n o r m a l level, suggesting that A C T I s e h a r a - 2 m i g h t n o t be s e c r e t e d f r o m the liver o r that it m a y b e r a p i d l y d e g r a d e d in the circulation.
Chandra T, Stackhouse R, Kidd VJ, Robson KJ, Woo SL (1983) Sequence homology between human c~l-antichymotrypsin, ulantitrypsin, and antithrombin III. Biochemistry 22:5055-5061 Hudig D, Haverty T, Fulcher C, Redelman D, Mendelson J (1981) Inhibition of human natural cytotoxicity by macromolecular antiproteases. J Immunol 126:1569-1574 Matsumoto M, Tsuda M, Kusumi T, Takada S, Shimamura T, Katsunuma T (1981) Enhancement by c~-l-antichymotrypsin of antibody response in vivo. Biochem Biophys Res Commun 100 : 478-482 Rubin H, Wang ZM, Nickbarg EB, McLarney S, Naidoo N, Shoenberger OL, Johnson JL, Cooperman BS (1990) Cloning, expression, purification and biological activity of recombinant native and variant human ~.l-antichymotrypsins. J Biol Chem 265:1199-1207 Takada S, Tsuda M, Yamamura M. Katsunuma T (1988) Effect of alpha-l-antichymotrypsin on activity of DNA primase isolated from human stomach adenocarcinoma cells. Biochem Int 16: 949-954 Travis J, Bowen J, Baugh R (1978) Human cq-antichymotrypsin: purification and properties. Biochemistry 17:5651-5656 Tsuda M, Ohkubo T, Kamiguchi H, Suzuki K, Nakasaki H, Mitomi T, Katsunuma T (1982) Purification, properties and identification of a serum DNA binding protein (64DP) and its microheterogeneity. Tokai J Exp Clin Med 7:201-211 Tsuda M, Masuyama M, Katsunuma T (1986) Inhibition of human DNA polymerase a by a~-antichymotrypsin. Cancer Res 46: 6139-6142 Tsuda M, Sei Y, Yamamura M, Yamamoto M, Shinohara Y (i992) Detection of a new mutant a-l-antichymotrypsin in patients with occlusive-cerebrovascular disease. FEBS LETT 304 : 66-68
