Title: Novel splice site mutation in the LIPH gene in a patient with autosomal recessive woolly hair/hypotrichosis: Case report and published work review Mizukami Y, Hayashi R, Tsuruta D, Shimomura Y, Sugawara K Ref: J Dermatol, 45:613, 2018 : PubMed
Autosomal recessive woolly hair is a relatively rare hereditary hair disorder characterized by sparse, short, curly hair. This condition is known to be caused by mutations in the LIPH gene, LPAR6 gene or KRT25 gene. In the Japanese population, most patients with autosomal recessive woolly hair carry one of two founder mutations in the LIPH gene, c.736T>A (p.Cys246Ser) or c.742C>A (p.His248Asn). However, occasionally, individuals with this condition carry compound heterozygous mutations, typically one founder mutation and another mutation. In this study, we describe a patient with a compound heterozygous mutation in the LIPH gene at c.736T>A and c.1095-3C>G. The latter mutation created a novel splice site. This was the fourth splice site mutation to be described in the LIPH gene. Furthermore, we performed an in vitro transcription assay in cultured cells, and demonstrated that the c.1095-3C>G mutation led to a frame-shift, which created a premature termination codon at the protein level (p.Glu366Ilefs*7). Finally, we summarized the mutations previously reported for the LIPH gene. Our findings provide further clues as to the molecular basis of autosomal recessive woolly hair.
The microminipig is one of the smallest minipigs that has emerged as a possible experimental animal model, because it shares many anatomical and/or physiological similarities with humans, including the coronary artery distribution in the heart, the digestive physiology, the kidney size and its structure, and so on. However, information on gene expression profiles, including those on drug-metabolizing phase I and II enzymes, in the microminipig is limited. Therefore, the aim of the present study was to identify transcripts in microminipig livers and to determine gene expression profiles. De novo assembly and expression analyses of microminipig transcripts were conducted with liver samples from three male and three female microminipigs using parallel long-read and short-read sequencing technologies. After unique sequences had been automatically aligned by assembling software, the mean contig length of 50843 transcripts was 707 bp. The expression profiles of cytochrome P450 (P450) 1A2, 2C, 2E1 and 3A genes in livers in microminipigs were similar to those in humans. Liver carboxylesterase (CES) precursor, liver CES-like, UDP-glucuronosyltransferase (UGT) 2C1-like, amine sulfotransferase (SULT)-like, N-acetyltransferases (NAT8) and glutathione S-transferase (GST) A2 genes, which are relatively unknown genes in pigs and/or humans, were expressed strongly. Furthermore, no significant gender differences were observed in the gene expression profiles of phase I enzymes, whereas UGT2B17, SULT1E1, SULT2A1, amine SULT-like, NAT8 and GSTT4 genes were different between males and females among phase II enzyme genes under the present sample conditions. These results provide a foundation for mechanistic studies and the use of microminipigs as model animals for drug development in the future. Copyright (c) 2016 John Wiley & Sons, Ltd.
Title: Analysis of unique mutations in the LPAR6 gene identified in a Japanese family with autosomal recessive woolly hair/hypotrichosis: Establishment of a useful assay system for LPA6 Hayashi R, Inoue A, Suga Y, Aoki J, Shimomura Y Ref: J Dermatol Sci, 78:197, 2015 : PubMed
BACKGROUND: Woolly hair (WH) is a hair shaft anomaly characterized by tightly-curled hair and is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal dominant or recessive inheritance. The autosomal recessive form of WH (ARWH) is caused by mutations in either lipase H (LIPH) or lysophosphatidic acid receptor 6 (LPAR6) gene, encoding an LPA-producing enzyme PA-PLA1alpha and an LPA receptor LPA6, respectively. OBJECTIVE: To define the molecular basis of ARWH/hypotrichosis in a Japanese family. METHODS: We performed mutational analysis of candidate genes and a series of expression and in vitro functional analyses, which we improved in this study, to determine the consequences resulting from the mutations identified in the family. RESULTS: Novel compound heterozygous LPAR6 mutations were identified in the patient. One was a nonsense mutation c.756T>A (p.Tyr252*); the other was a large insertion mutation within the promoter region of LPAR6. Expression studies detected LPAR6 mRNA only from the c.756T>A allele in the patient's hair follicles, suggesting that the insertion in the other allele disrupted the LPAR6 promoter and thus led to a failure of transcription. Furthermore, an improved LPA6 functional assay developed in this study demonstrated aberrant expression and a subsequent loss of function of the p.Tyr252*-mutant protein. CONCLUSION: Through establishing a useful assay system for LPA6, our results further underscore the crucial roles of LPAR6 in hair follicle development and hair growth in humans at molecular levels.
Title: Identification of a novel mutation, c.686delAins18 (p.Asp229Glyfs*22), in the LIPH gene as a compound heterozygote with c.736T>A (p.Cys246Ser) in autosomal recessive woolly hair/hypotrichosis Ito T, Shimomura Y, Hayashi R, Tokura Y Ref: J Dermatol, 42:752, 2015 : PubMed
Title: Compound heterozygous mutations in two distinct catalytic residues of the LIPH gene underlie autosomal recessive woolly hair in a Japanese family Hayashi R, Akasaka T, Ito M, Shimomura Y Ref: J Dermatol, 41:937, 2014 : PubMed
Title: Expression studies of a novel splice site mutation in the LIPH gene identified in a Japanese patient with autosomal recessive woolly hair Hayashi R, Inui S, Farooq M, Ito M, Shimomura Y Ref: J Dermatol, 41:890, 2014 : PubMed
Autosomal recessive woolly hair (ARWH) is characterized by short and tightly curled scalp hair without any obvious complications. The disease is known to be caused by either lipase H (LIPH) or LPAR6 genes. Proteins encoded by these two genes are closely related to each other in a lipid-signaling pathway that is believed to play crucial roles in hair follicle development and hair growth. In the Japanese population, most affected individuals with ARWH have been shown to carry two prevalent founder mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.742C>A (p.His248Asn), while other LIPH mutations have been occasionally identified. In this study, we analyzed a Japanese patient with ARWH, and identified compound heterozygous mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.982+5G>T. The latter one was a novel splice site mutation in intron 7. Expression studies using blood-derived RNA from the patient detected the LIPH transcript from the c.736T>A mutant allele, but not from the c.982+5G>T mutant allele. Furthermore, in vitro transcription assay in cultured cells showed that the mutation c.982+5G>T caused an aberrant splicing event, leading to a frame-shift and a premature termination codon (p.Met328Serfs*41). To the best of our knowledge, this is the second splice site mutation in the LIPH gene, and our findings further expand the spectrum of the LIPH mutations underlying ARWH.
To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.
Title: Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction Mima J, Hayashida M, Fujii T, Narita Y, Hayashi R, Ueda M, Hata Y Ref: Journal of Molecular Biology, 346:1323, 2005 : PubMed
Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.
Title: Crystallization and preliminary X-ray analysis of carboxypeptidase Y inhibitor IC complexed with the cognate proteinase Mima J, Hayashida M, Fujii T, Hata Y, Hayashi R, Ueda M Ref: Acta Crystallographica D Biol Crystallogr, 60:1622, 2004 : PubMed
Carboxypeptidase Y (CPY) inhibitor I(C) is a naturally occurring serine carboxypeptidase inhibitor from Saccharomyces cerevisiae, the sequence of which is not homologous with any other known proteinase inhibitor and is classified as the phosphatidylethanolamine-binding protein (PEBP). I(C) has been crystallized in complex with the deglycosylated form of CPY by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant. The crystals of the complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 81.13, b = 186.6, c = 65.14 A. Diffraction data were collected to 2.7 A resolution. Structure determination of the complex is in progress by the molecular-replacement method using the structure of CPY as a search model.
Title: The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase. Implications for the biological roles of the phosphatidylethanolamine-binding protein Mima J, Narita Y, Chiba H, Hayashi R Ref: Journal of Biological Chemistry, 278:29792, 2003 : PubMed
The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs). In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated. The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site. IC is able to form the complex with diisopropyl fluorophosphate-modified CPY. Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY. These findings demonstrate the multiple site binding of IC with CPY. Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs.
Title: Increased proteolytic susceptibility of carboxypeptidase Y caused by modification of the disulfide zipper Maki T, Kozawa H, Mima J, Ueno H, Hayashi R Ref: Biosci Biotechnol Biochem, 66:1393, 2002 : PubMed
To investigate the structural importance of a "disulfide zipper" motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility.
Title: Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y Mima J, Jung G, Onizuka T, Ueno H, Hayashi R Ref: European Journal of Biochemistry, 269:3220, 2002 : PubMed
Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.
Title: N-terminal acetyl group is essential for the inhibitory function of carboxypeptidase Y inhibitor (I(C)) Mima J, Kondo T, Hayashi R Ref: FEBS Letters, 532:207, 2002 : PubMed
Carboxypeptidase Y (CPY) inhibitor, I(C), a yeast cytoplasmic inhibitor in which the N-terminal amino acid is acetylated, was expressed in Escherichia coli and produced as an unacetylated form of I(C) (unaI(C)). Circular dichroism and fluorescence measurements showed that unaI(C) and I(C) were structurally identical and produce identical complexes with CPY. However, the K(i) values for unaI(C) for anilidase and peptidase activity of CPY were much larger, by 700- and 60-fold, respectively, than those of I(C). The reactivities of phenylmethylsulfonyl fluoride and p-chloromercuribenzoic acid toward the CPY-unaI(C) complex were considerably higher than those toward the CPY-I(C) complex. Thus, the N-terminal acetyl group of I(C) is essential for achieving a tight interaction with CPY and for its complete inactivation.
Title: Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae Mima J, Suzuki H, Takahashi M, Hayashi R Ref: J Biochem, 132:967, 2002 : PubMed
Carboxypeptidase Y (CPY) inhibitor, I(C), a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1. The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular weight of 24,398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy. The acetylated N-terminal methionine residue is the sole posttranslational modification. I(C) specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (K(i)) of approximately 1.0 x 10(-9) M. The chemical modification of I(C) with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure. The formation of a complex of I(C) with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-I(C) complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex.
Title: Carboxypeptidase Y: structural basis for protein sorting and catalytic triad Jung G, Ueno H, Hayashi R Ref: J Biochem, 126:1, 1999 : PubMed
A yeast vacuolar protease, carboxypeptidase Y (CPY), is known to be involved in the C-terminal processing of peptides and proteins; however, its real function remains unclear. The CPY biosynthetic pathway has been used as a model system for protein sorting in eukaryotes. CPY is synthesized as a prepro-form that travels through the ER and Golgi to its final destination in vacuoles. In the course of studies on the transport mechanism of CPY, various post-translational events have been identified, e.g. carbohydrate modification and cleavage of the pre-segments. In addition, sorting signals and various sorting vehicles, similar to those found in higher eukaryotic cells, have been found. The catalytic triad in the active site of CPY makes this enzyme a serine protease. A unique feature distinguishing CPY from other serine proteases is its wide pH optimum, in particular its high activity at acidic pH. Several structural properties which might contribute to this unique feature exist such as a conserved free cysteine residue in the S1 substrate binding pocket, a recognition site for a C-terminal carboxyl group, and a disulfide zipper motif. The structural bases in CPY functions are discussed in this article.
Title: Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y Jung G, Ueno H, Hayashi R, Liao TH Ref: Protein Science, 4:2433, 1995 : PubMed
The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.
Title: Purification and characterization of lipoprotein lipase from human postheparin plasma and its comparison with purified bovine milk lipoprotein lipase Hayashi R, Tajima S, Yamamoto A Ref: J Biochem, 100:319, 1986 : PubMed
Lipoprotein lipase [EC 3.1.1.34, LpL] was purified from human postheparin plasma (PHP) almost to homogeneity (a 210,000-fold purification) using columns of heparin-Sepharose, hydroxylapatite, and concanavalin A-Sepharose, and its properties were compared with the purified bovine milk LpL. The specific activity of the PHP-LpL was 26 mmol free fatty acids (FFA)/h/mg of protein at 37 degrees C; close to that of bovine milk LpL (35 mmol FFA/h/mg). For both enzyme preparations, the pH optimum (about 8.7) and the inhibition by sodium chloride were almost the same. The apparent Michaelis constants were also similar; 2.5 mM for human PHP-LpL and 2.1 mM for bovine milk LpL. The apparent molecular weight of the purified human PHP-LpL was 58,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, slightly larger than that of the bovine milk LpL (56,000). Although the amino acid composition of the two LpL preparations had only slight differences, antibody raised against bovine milk LpL cross-reacted very weakly with purified human PHP-LpL. With 1% bovine serum albumin, bovine milk LpL was highly stable, but the human PHP-LpL was unstable; it lost 60% of its activity within 60 min at 0 degrees C. In the absence of apolipoprotein C-II (apo C-II), the activity of human PHP-LpL was very weak. However, human PHP-LpL was activated by apo C-II more strongly than bovine milk LpL; the fold activation of human PHP-LpL by apo C-II was 7-8 times that of bovine milk LpL. The apparent Km value of human PHP-LpL for apo C-II (1.00 +/- 0.58 microM) was larger than that of bovine milk LpL (0.15 +/- 0.03 microM).
Title: Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue Hayashi R, Bai Y, Hata T Ref: J Biochem, 77:1313, 1975 : PubMed
The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.
Title: Serine at the active center of yeast carboxypeptidase Hayashi R, Moore S, Stein WH Ref: Journal of Biological Chemistry, 248:8366, 1973 : PubMed
