Title: The β9 Loop Domain of PA-PLA1α Has a Crucial Role in Autosomal Recessive Woolly Hair/Hypotrichosis
Abstract: amino acid alkaline phosphatase–tagged transforming growth factor-α free fatty acid lysophosphatidic acid phosphatidic acid membrane-associated phosphatidic acid–preferring phospholipase A1α phospholipase A1 phosphatidylserine phosphatidylserine-specific phospholipase A1 triacylglycerol wild type TO THE EDITOR Membrane-associated phosphatidic acid–preferring phospholipase A1α (PA-PLA1α, also known as mPA-PLA1α and lipase H (LIPH)) is an enzyme known to hydrolyze phosphatidic acid (PA) into 2-acyl lysophosphatidic acid (LPA) and free fatty acid (FFA; Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar; Hiramatsu et al., 2003Hiramatsu T. Sonoda H. Takanezawa Y. et al.Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.J Biol Chem. 2003; 278: 49438-49447Crossref PubMed Scopus (82) Google Scholar). It has recently been shown that LPA, which is produced by PA-PLA1α, works as a ligand for its receptor LPA6, also known as P2Y5, and is speculated to regulate the proliferation and differentiation of inner root sheath cells of hair follicles (Inoue et al., 2011Inoue A. Arima N. Ishiguro J. et al.LPA-producing enzyme PA-PLA(1)α regulates hair follicle development by modulating EGFR signalling.EMBO J. 2011; 30: 4248-4260Crossref PubMed Scopus (101) Google Scholar). Recently, LIPH (MIM# 607365) and LPAR6 (MIM# 609239), which encode PA-PLA1α and LPA6, respectively, were identified as causative genes for autosomal recessive woolly hair with associated hypotrichosis (ARWH/H; MIM# 604379, 278150; Kazantseva et al., 2006Kazantseva A. Goltsov A. Zinchenko R. et al.Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.Science. 2006; 314: 982-985Crossref PubMed Scopus (161) Google Scholar; Pasternack et al., 2008Pasternack S.M. von Kugelgen I. Aboud K.A. et al.G protein-coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth.Nat Genet. 2008; 40: 329-334Crossref PubMed Scopus (329) Google Scholar; Shimomura et al., 2008Shimomura Y. Wajid M. Ishii Y. et al.Disruption of P2RY5, an orphan G protein-coupled receptor, underlies autosomal recessive woolly hair.Nat Genet. 2008; 40: 335-339Crossref PubMed Scopus (176) Google Scholar). To our knowledge, we identified a previously unreported heterozygous missense mutation at the β9 loop domain of PA-PLA1α, which is considered a crucial structure for substrate recognition (Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar; Aoki et al., 2007Aoki J. Inoue A. Makide K. et al.Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family.Biochimie. 2007; 89: 197-204Crossref PubMed Scopus (121) Google Scholar) in this study. To clarify the role of the β9 loop domain, the hydrolytic activity and LPA6 activation ability of mutant PA-PLA1α were evaluated. A 3-year-old Japanese girl was seen at our hospital with woolly and sparse hair on her scalp without other abnormalities (Figure 1a). Her eyebrows and eyelashes were slightly sparse. Her parents were unrelated and nonconsanguineous, and had normal hair. Blood samples were collected for DNA extraction in accordance with standard methods, and an LIPH mutation search was performed as previously reported (Shimomura et al., 2009Shimomura Y. Wajid M. Petukhova L. et al.Mutations in the lipase H gene underlie autosomal recessive woolly hair/hypotrichosis.J Invest Dermatol. 2009; 129: 622-628Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Direct sequencing analysis of all exons and intron–exon boundaries of the LIPH revealed that the patient was compound heterozygous for the two missense mutations c.619G>C (p.Asp207His) and c.742C>A (p.His248Asn), which were segregated from her mother and father, respectively (Figure 1b). These mutations were verified by restriction enzyme digestions of the PCR products by Hpy188I and mutant allele–specific amplification analysis, respectively (Supplementary Figure S1 online). p.His248Asn, one amino acid (a.a.) of the catalytic triad, is known as a prevalent pathogenic mutation in the Japanese population (Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar). Download .pdf (.35 MB) Help with pdf files Supplementary Figures To the best of our knowledge, c.619G>C (p.Asp207His) in LIPH is a previously unreported mutation and was not found in alleles from 100 normal unrelated individuals. Asp207 residue of PA-PLA1α is conserved among diverse species, suggesting that Asp207 may have a critical role in enzyme activity (Supplementary Figure 2a online). To assess the role of Asp207 as a candidate for the ARWH/H, two distinct in vitro functional analyses were performed: for hydrolytic activity and for LPA6 activation ability of PA-PLA1α (Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar). To investigate the molecular defects underlying the mutation, we synthesized p.Asp207His PA-PLA1α expression constructs and compared the mutant protein expression with wild-type (WT) and with p.Ser154Ala, which was known as a loss-of-function mutation (Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar; Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar). Immunoblot analysis using anti-PA-PLA1α monoclonal antibody revealed that the transfection of p.Asp207His constructs into HEK293 cells resulted in the secretion of the 55-kDa mutant PA-PLA1α at levels similar to those of WT and p.Ser154Ala (Figure 2a; Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar). In addition, the same amounts of mutant PA-PLA1α proteins were also recovered from the cell lysate (data not shown). These results indicate that there was no significant difference in protein amount between WT and mutant PA-PLA1α. Hydrolysis activity was determined by measuring FFA, which was concurrently produced from PA by PA-PLA1α. Briefly, we added the supernatant from HEK293 cells transfected with WT, p.Ser154Ala, or p.Asp207His PA-PLA1α to a medium containing 400μM PA. After 3hours of incubation at 37°C, the amount of oleic acids, one kind of FFA, was measured using a NEFA C-Test Wako test kit (Wako Chemicals, Osaka, Japan; Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar). The quantities of oleic acids produced by the p.Asp207His PA-PLA1α were markedly lower than those for the WT; however, the p.Asp207His PA-PLA1α slightly retained the function of hydrolytic activity relative to mock and p.Ser154Ala (Figure 2b). To perform the LPA6 activation ability assay, alkaline phosphatase–tagged transforming growth factor-α (AP-TGFα; kindly provided by Dr Higashiyama, Ehime University, Japan; Tokumaru et al., 2000Tokumaru S. Higashiyama S. Endo T. et al.Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.J Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar), recombinant LPA6, and PA-PLA1α constructs (WT, p.Ser154Ala or p.Asp207His) were cotransfected to HEK293 cells. To measure the LPA6 activation potency of mutant PA-PLA1α, AP-TGFα release into conditioned media via a disintegrin and metalloprotease, which was triggered by the activation of LPA6, was quantified using p-NPP as a substrate for AP (Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar; Inoue et al., 2011Inoue A. Arima N. Ishiguro J. et al.LPA-producing enzyme PA-PLA(1)α regulates hair follicle development by modulating EGFR signalling.EMBO J. 2011; 30: 4248-4260Crossref PubMed Scopus (101) Google Scholar). As previously reported, the free AP-TGFα from the LPA6-untransfected (LPA6-) cells expressing the WT PA-PLA1α was more abundant than that from the LPA6- cells transfected with mock PA-PLA1α, which indicated that the HEK293 cells had the ability to shed AP-TGFα mediated by intrinsic LPA receptor at some level (Figure 2c; Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar; Inoue et al., 2011Inoue A. Arima N. Ishiguro J. et al.LPA-producing enzyme PA-PLA(1)α regulates hair follicle development by modulating EGFR signalling.EMBO J. 2011; 30: 4248-4260Crossref PubMed Scopus (101) Google Scholar). AP-TGFα release from LPA6-transfected (LPA6+) cells expressing the WT PA-PLA1α was remarkably increased compared with mock or mutant PA-PLA1α. There were no significant differences between the data obtained with cells expressing the mutant and mock PA-PLA1α (Figure 2c). These data indicate that the p.Asp207His PA-PLA1α results in the complete loss of LPA6 activation activity. Despite the remaining slight hydrolytic activity, there was complete loss of LPA6 activation ability and no significant difference in clinical features between this patient and other patients with LIPH mutations who were revealed as having complete loss of hydrolytic activity (Shinkuma et al., 2010Shinkuma S. Akiyama M. Inoue A. et al.Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.Hum Mutat. 2010; 31: 602-610PubMed Google Scholar). These findings suggest that the p.Asp207His PA-PLA1α retained slight hydrolytic activity, which was insufficient to activate LPA6 and possibly leading to woolly hair. Phospholipase A1 (PLA1) is an enzyme that hydrolyzes ester bonds of phospholipids (Aoki, 2004Aoki J. Mechanisms of lysophosphatidic acid production.Semin Cell Dev Biol. 2004; 15: 477-489Crossref PubMed Scopus (246) Google Scholar). PLA1, such as phosphatidylserine (PS)-specific PLA1 (PS-PLA1), PA-PLA1α, and PA-PLA1β, form a subfamily in the pancreatic lipase gene family (Sato et al., 1997Sato T. Aoki J. Nagai Y. et al.Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family.J Biol Chem. 1997; 272: 2192-2198Crossref PubMed Scopus (120) Google Scholar; Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar; Hiramatsu et al., 2003Hiramatsu T. Sonoda H. Takanezawa Y. et al.Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.J Biol Chem. 2003; 278: 49438-49447Crossref PubMed Scopus (82) Google Scholar). These PLA1 enzymes distinctly differ from other lipases in that PLA1 enzymes do not hydrolyze triacylglycerol (TG) and that they show strict substrate specificities and act specifically on PS and PA, respectively (Sato et al., 1997Sato T. Aoki J. Nagai Y. et al.Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family.J Biol Chem. 1997; 272: 2192-2198Crossref PubMed Scopus (120) Google Scholar; Sonoda et al., 2002Sonoda H. Aoki J. Hiramatsu T. et al.A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.J Biol Chem. 2002; 277: 34254-34263Crossref PubMed Scopus (165) Google Scholar; Hiramatsu et al., 2003Hiramatsu T. Sonoda H. Takanezawa Y. et al.Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.J Biol Chem. 2003; 278: 49438-49447Crossref PubMed Scopus (82) Google Scholar). The lipase family has three a.a. residues that form the putative catalytic triad and has three surface loops called the lid, the β5 loop, and the β9 loop that cover the active site (Winkler et al., 1990Winkler F.K. D’Arcy A. Hunziker W. Structure of human pancreatic lipase.Nature. 1990; 343: 771-774Crossref PubMed Scopus (1033) Google Scholar; Carriere et al., 1998Carriere F. Withers-Martinez C. van Tilbeurgh H. et al.Structural basis for the substrate selectivity of pancreatic lipases and some related proteins.Biochim Biophys Acta. 1998; 1376: 417-432Crossref PubMed Scopus (119) Google Scholar; Aoki et al., 2007Aoki J. Inoue A. Makide K. et al.Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family.Biochimie. 2007; 89: 197-204Crossref PubMed Scopus (121) Google Scholar). A comparison of the a.a. sequences of PLA1 and TG lipases, such as pancreatic lipase and lipoprotein lipase, revealed that PLA1 enzymes have shorter lid and β9 loop than TG lipases (PLA1: 12 a.a. and 13 a.a.; TG lipases: 22–23 a.a. and 18–19 a.a., respectively; Supplementary Figure 2b online; Aoki et al., 2007Aoki J. Inoue A. Makide K. et al.Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family.Biochimie. 2007; 89: 197-204Crossref PubMed Scopus (121) Google Scholar). Therefore, the lid, the β5 loop, and the β9 loop are implicated in substrate specificity. In this study, we identified the p.Asp207His mutation at the β9-loop domain of PA-PLA1α in the ARWH/H patient, and clarified that the mutant showed substantial abolition of hydrolytic activity and had no LPA6 activation ability. These results confirm that the β9-loop domain of PA-PLA1α has a crucial role in enzyme activity. We thank the patient and her parents for their generous cooperation and Ms Junko Tamba and Ms Yui Saito for their technical assistance. The family gave written informed consent. The medical ethics committee of Hokkaido University approved all the described studies. The study was conducted according to the Declaration of Helsinki Principles. This work was supported by the Naito Foundation. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid