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Isolation and Expression of a Malassezia globosa Lipase Gene, LIP1 Yvonne M. DeAngelis1, Charles W. Saunders1, Kevin R. Johnstone1, Nancy L. Reeder1, Christal G. Coleman2, Joseph R. Kaczvinsky Jr1, Celeste Gale1, Richard Walter1, Marlene Mekel1, Martin P. Lacey1, Thomas W. Keough1, Angela Fieno1, Raymond A. Grant1, Bill Begley1, Yiping Sun1, Gary Fuentes1, R. Scott Youngquist1, Jun Xu1 and Thomas L. Dawson Jr1

Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology. Journal of Investigative Dermatology (2007) 127, 2138–2146; doi:10.1038/sj.jid.5700844; published online 26 April 2007

INTRODUCTION rheic dermatitis because of their shared symptoms, primarily Dandruff and seborrheic dermatitis (D/SD) are common skin excessive epidermal cell proliferation and pruritis. Most disorders associated with Malassezia, with dandruff occurring recent data indicate that dandruff is a mild form of seborrheic in at least 40–50% and seborrheic dermatitis in 1–3% of the dermatitis (Faergemann, 2000; Ashbee and Evans, 2002; general population of immunocompetent adults (Warner Gupta et al., 2004a). et al., 2001; Ashbee and Evans, 2002). The incidence of SD D/SD are usually associated with the presence of Malasse- is much higher in immunocompromised patients, especially zia and can be effectively treated with a variety of antifungal AIDS patients, ranging from 30–33% (Farthing et al., 1985; agents (Shuster, 1984; Schwartz et al., 2004). Recent reports Smith et al., 1994). D/SD have received recent attention, as have described the Malassezia species found on the scalp. their presence leads to a negative social impact (Warner Using culture-based methods, Midgley (2000) found M. et al., 2001). Dandruff is generally categorized with sebor- globosa as the most abundant species, with lesser amounts of M. sympodialis, M. restricta, and M. slooffiae. Gue´ho et al. (1996) Aspiroz et al. (1999), also used culture-based methods 1 The Procter & Gamble Company, Miami Valley Innovation Center, and found M. restricta and M. globosa to be the most common Cincinnati, Ohio, USA and 2Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, NY, USA isolates, with a lesser amount of M. sympodialis. Using PCR- Correspondence: Dr Thomas L. Dawson Jr, Beauty Technology Division, based methods, Gemmer et al. (2002) found primarily M. The Procter & Gamble Company, 11810 East Miami River Road, Cincinnati, restricta and M. globosa.However,M. yamatoensis, M. furfur, Ohio 45252, USA. E-mail: [email protected] M. sympodialis, M. obtusa,andM. slooffiae have also been Abbreviations: BSTFA, bis(trimethylsilyl)triflouroacetamide; D/SD, dandruff isolated from the skin of D/SD patients (Gupta et al., 2000; and seborrheic dermatitis; dPBS, Dulbecco’s phosphate-buffered saline; ESI Nakabayashi et al., 2000; Sugita et al., 2004). MS MS, electrospray ionization tandem mass spectrometry; FID, flame ionization detector; FFA, free fatty acid; GC, gas chromatography; MALDI Fungi in the genus Malassezia (formerly Pityrosporum) are PSD, matrix-assisted laser desorption ionization post source decay; MES, associated with several human diseases and conditions, 2-morpholinoethanesulfonic acid; Page, polyacrylamide gel electrophoresis; including pityriasis versicolor, atopic dermatitis, seborrheic PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; RACE, dermatitis, folliculitis, and dandruff (Erchiga and Florencio, rapid amplification of cDNA ends; RT–PCR, reverse transcription-PCR; TMSI, N-trimethylsilylimidazole. 2002; Gupta et al., 2004a). The genus Malassezia has been Received 10 October 2006; revised 12 January 2007; accepted 20 January shown to comprise at least 10 species, based on ribosomal 2007; published online 26 April 2007 DNA characterization (Guillot and Gue´ho, 1995; Sugita

2138 Journal of Investigative Dermatology (2007), Volume 127 & 2007 The Society for Investigative Dermatology YM DeAngelis et al. Malassezia globosa Lipase Gene Cloning and Expression

et al., 2002, 2003; Gupta et al., 2004a) and their ability to 15 grow in certain media (Mayser et al., 1997). There is considerable interest in correlating the presence of individual species of Malassezia with effects on human skin health 10 (Gue´ho et al., 1998; Ashbee and Evans, 2002; Faergemann, 2002; Gemmer et al., 2002; Gupta et al., 2004a, b). The role of the fungus in D/SD has been attributed to a 5 variety of factors, including (1) induction of an immune response (Kikuchi et al., 1989), (2) fungal lipase that Lipase activity pmol/
g/minute generates irritating fatty acids (Priestley and Savin, 1976), (3) lipid peroxidase that generates irritating lipid metabolites

(Hay and Graham-Brown, 1997), and (4) a toxin (Pie´rard- M.furfur M.obtusa M.globosa M.restricta Franchimont et al., 2000). These concepts have not been M.slooffiae tested directly, owing to both a dearth of biochemical M.sympodialis M.pachydermatis understanding of Malassezia and the lack of pure molecules whose effects could be tested. For example, there are no Figure 1. Lipase activity was detected in four different Malassezia species. Values represent pmoles of p-nitrophenol released per microgram total cloned M. globosa or restricta genes suspected to be involved cell protein per minute incubation. The reaction was carried out as described in any of these proposed mechanisms of pathology. in the Materials and Methods section except that that the incubation was Malassezia lipases may be involved in D/SD. The human for 15 minutes at 301 and 1.3% Triton X-100 was included in the assay skin-associated Malassezia all require lipid for growth in vitro buffer. Error bars represent standard error of three replicate experiments. (Midgley, 2000; Batra et al., 2005). It seems likely that these Malassezia strains reported here are M. globosa CBS 7966, M. furfur CBS fungi would also require lipid for growth on human skin and 7982, M. obtusa CBS 7968, M. restricta CBS 7877, M. slooffiae CBS 7956, that lipase-mediated digestion of sebum glycerides would be M. sympodialis 7977, and M. pachydermatis ATCC 74522. the first step in lipid consumption. Lipase activity has been reported from Malassezia furfur, with the activity found only cells was measured using p-nitrophenyl oleate as a substrate. in the insoluble cell debris fraction (Ran et al., 1993), in both Lipase activity was readily detected in M. pachydermatis, M. the culture supernatant and cell homogenate (Plotkin et al., furfur, M. globosa, and M. sympodialis (Figure 1). In contrast, 1996), in the culture supernatant (Mancianti et al., 2000), and no lipase activity was detected with M. restricta, M. slooffiae, on agar plates containing M. furfur (Muhsin et al., 1997). and M. obtusa. However, the lack of lipase activity was Lipase activity has also been reported from the culture possibly an artifact of in vitro culture or assay conditions and supernatant of M. pachydermatis (Mancianti et al., 2000). may not represent lipase production when these organisms Recently, lipase genes have been cloned from M. furfur are present on host skin. (Brunke and Hube, 2006) and M. pachydermatis (Shibata et al., 2006). However, M. furfur and M. pachydermatis are M. globosa lipase not known to colonize human scalp (Aspiroz et al., 1999, There was no detectable lipase activity in the supernatant of Gemmer et al., 2002), so these are unlikely to play a role in M. globosa cultures. However, upon extracting intact cells dandruff. There are no reports of lipase activity from other with 50 mM Tris–HCl (pH 8), 100 mM NaCl, lipase activity was Malassezia species. readily detected. This was demonstrated in several different Our goal was to identify lipase activity from a scalp fungus lipase assays. In one assay, based on gas chromatography with a role in dandruff. We began by surveying the lipase analysis of fatty acids released by this extracted lipase, the M. activity of seven species of Malassezia. Of the two Malassezia globosa lipase hydrolyzed diolein, generating oleic acid species most commonly found on the scalp, only M. globosa (Figure 2a). However, the lipase showed no activity against produced measurable lipase activity in culture. We purified a triolein (Figure 2a and b). In contrast, when M. globosa cells lipase from M. globosa and cloned the corresponding gene, were used as the lipase source, triolein hydrolysis was which we have named LIP1 (Genebank accession No. observed (Figure 2b). DD210404). The lipase gene expression was regulated under In a complementary assay, a colorimetric method was differing culture conditions and was detected on human used to detect glycerol, the other product of lipase action. scalps, as revealed by reverse transcription-PCR (RT–PCR). The The extracted lipase hydrolyzed olive oil, diolein, and partially purified lipase was able to hydrolyze diglycerides but monoolein but had little or no effect on triolein (Figure 3). not triglycerides. Thus, this report describes the first identifica- In contrast, intact M. globosa cells hydrolyzed all of these tion and characterization of the Malassezia-mediated lipid substrates. The lipase was inactivated by boiling for metabolic process likely involved in the etiology of the most 10 minutes, and a microbial lipase inhibitor, fospirate (Weeks common hyperproliferative skin disorders, D/SD. et al., 1977), prevented the olive oil hydrolysis by both the extracted lipase and cells. In addition to the lipase activity at RESULTS pH 5.5, both extract and cells were active in phosphate- Lipase activity from different malassezia species buffered saline, with 69 and 57% of the full activity, Lipase production was evaluated from a representative strain respectively. However, with both extract and cells, o3% of of several species of Malassezia. The lipase activity of washed the activity was present at pH 8.0.

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a Lipase purification The lipase was further purified, using hydrolysis of p- nitrophenyl oleate as an assay. After separation by anion OA exchange liquid chromatography, the lipase-containing frac- MO tions were analyzed using native gel electrophoresis (Fig- DO ure 4). Gel fractions were assayed for lipase activity, which TO was mostly found in gel fractions 8 and 9. These fractions were pooled and analyzed by SDS gel electrophoresis, where GC peak area counts the major band was a 32 kDa protein. Control M.globosa Isolated cells lipase Lipase protein sequence b To generate N-terminal sequence of the 32 kDa protein, an SDS gel was blotted onto polyvinylidene difluoride and stained with Coomassie blue. The 32 kDa band was excised OA from the blot and characterized by Edman sequencing. A 22- MO amino-acid sequence was determined. Internal protein DO sequence was also determined for the gel-purified 32 kDa TO protein. The gel slices were digested with trypsin, and peptides were separated by HPLC and sequenced using a GC peak area counts combination of matrix-assisted laser desorption ionization Control M.globosa Isolated post source decay, nanospray MSMS, and Edman methods. cells lipase Lipase gene cloning Figure 2. Gas chromatography analysis reveals that isolated lipase is active Oligonucleotides were designed based on the peptide against diolein and not triolein. Extracted and cell-based lipase activity were compared, using (a) a triolein/diolein mixture and (b) triolein as a sequence and used in PCR to amplify a segment of the lipase substrate, with the values, on a linear scale, indicating the amount of oleic gene. To isolate additional lipase coding sequences, we used acid (OA), monoolein (MO), diolein (DO), and triolein (TO) present after 30 and 50 rapid amplification of cDNA ends (RACE) on cDNA, incubation. The composition of the substrate alone is indicated in the extending in each direction. To ensure that the apparently control samples at the left-hand side of the figure. overlapping gene segments were from the same gene, a single PCR was carried out using genomic DNA and oligonucleo- Extract Cells Recombinant LIP1 tides whose sequence matched nucleic acid sequence near 2.5 the distant ends of the RACE products. The resulting product 2 was cloned, sequenced, and named LIP1 (see Genbank 1.5 accession number DD210404). 1 The DNA sequence of the LIP1 segment indicated that the

OD 540 nm cloned gene encoded the 32 kDa protein that had been

Lipase activity 0.5 purified and that the protein was a lipase. First, the predicted translation product matched the sequence of several internal Olive oil DioleinTriolein Olive oil DioleinTriolein Olive oil DioleinTriolein peptides plus the N-terminal peptide. Further scrutiny of this Monoolein Monoolein Monoolein translation product revealed the amino-acid sequence

Olive oil+0.003% fospirate Olive oil+0.003% fospirate Olive oil+0.003% fospirate

Figure 3. LIP1 hydrolyzed diolein and monolein but not triolein. Values 1.2 represent the lipase activity in the glycerol detection assay with the indicated substrate and, where indicated, substrate plus inhibitor. For each of the

samples, a boiled lipase control was performed, and its OD540 value was 0.6 97.4 subtracted from the OD of the lipase test sample, with the difference in 66.2 540 45.0 OD540 recorded on the graph. Error bars indicate the standard deviations of Lipase activity the background-subtracted values, with 12 replicates of the samples 0.0 31.0 containing extract and cells and four replicates of the samples containing Fractions 45678910 1112 13 14 15 16 17 recombinant LIP1. 21.5

14.4 As little or no cell lysis was detected (as indicated by the small number of detectable proteins after SDS polyacryla- Native PAGE SDS PAGE mide gel electrophoresis of the extract, data not shown) under Figure 4. Lipase was isolated using native polyacrylamide gel lipase extraction conditions, the diglyceride-hydrolyzing electrophoresis. Native gel fractions were assayed for the presence of lipase, lipase activity was extracellular, perhaps loosely associated using p-nitrophenol oleate. Fractions 8 and 9 of the native gel were pooled with the cell wall. and analyzed by SDS polyacrylamide gel electrophoresis.

2140 Journal of Investigative Dermatology (2007), Volume 127 YM DeAngelis et al. Malassezia globosa Lipase Gene Cloning and Expression

a O

s O N O O NH H O

P O O NH O HN O

O

O2N b 45k

Avidin dimer Lipase 34k

23k Avidin

16k

Figure 5. A covalent lipase inhibitor was used to isolate LIP1. (a) Biotinylated lipase inhibitor. (b) SDS polyacrylamide gel electrophoresis of proteins recovered from avidin beads in the presence and absence of biotinylated lipase inhibitor. The size of molecular weight markers is indicated.

GHSLG, a peptide sequence found at the active site of many consensus N-linked glycosylation site. Note that the absence lipases (Pigne`de et al., 2000). The predicted LIP1-encoded of other lipases detectable in the gel (Figure 5b) does not protein sequence was searched against the Pfam database mean that other lipases are not present. Other lipases may (release 20, Bateman et al., 2004). The best match is the have failed to react with the substrate either due to poor lipase 3 protein family (lipase class 3, accession number: binding interactions or inaccessibility of the lipase, insuffi- PF01764) with a highly significant E value (3.7 Â 10À24). The cient amounts of the lipase may have been recovered after LIP1-encoded protein sequence was also used to search boiling, or additional lipases may have had the same against the GenBank NR database using BLASTP (Altschul electrophoretic mobility as another protein. et al., 1997). Of the top 20 hits (E value less than 2 Â 10À13, As further evidence that LIP1 encodes a lipase, the identity 431% and similarity 451%), 18 were annotated as recombinant LIP1 was produced in P. pastoris and shown lipases and two were annotated as hypothetical proteins. All to have lipase activity with similar substrate specificity as the the top 20 hits contain a lipase 3 family domain as well. M. globosa extract (Figure 3).

Confirmation that LIP1 encodes a lipase LIP1 cDNA expression is regulated in vitro A synthetic, irreversible lipase inhibitor (Figure 5a), cova- Expression levels were monitored at different stages of growth lently coupled to biotin (Deussen et al., 2000), was used to and with different fatty acid supplementation (Figure 6a). The isolate lipase from M. globosa cells. To demonstrate that the ratio of LIP1 to ACT1 mRNA was not obviously different as inhibitor could be useful for lipase isolation, a Thermomyces the cell culture became more confluent (Figure 6b). How- lanuginosa lipase was recovered after reacting with the ever, alteration of the sole available lipid source affected LIP1 inhibitor (data not shown). M. globosa cells were treated with expression. The M. globosa lipid requirement can be fulfilled the inhibitor, and covalently bound proteins were isolated with either free fatty acids (FFAs; we used an oleic acid and separated by SDS gel electrophoresis (Figure 5b). A sample comprising B74% oleic acids with the remainder 32 kDa protein was observed. The 32 kDa protein was primarily other FFAs, as measured by thin layer chromato- digested with trypsin and eluted from the gel. The sequence graphy (NuChek Prep, Elysian, MN; data not shown) or of the trypsin-generated fragments matched that predicted for Tween-40s, a poly-sorbitan with esterified fatty acids. M. the translation product of LIP1, with over 81% of the amino- globosa was cultured in mDixon medium modified so that acid sequence observed. The only hypothetical tryptic only one of these lipid sources was present (Materials and fragments not observed were (1) lower than 600 Da, below Methods section). LIP1 was more highly expressed in the the mass range of the experiment, or (2) the tryptic peptide Tween-40s-containing medium than in the oleic acid- ALGYQHPSDYVWIYPGNSTSAK which contains an internal containing medium (Figure 6a). This result indicates regula-

www.jidonline.org 2141 YM DeAngelis et al. Malassezia globosa Lipase Gene Cloning and Expression

ab sympodialis. Among the species for which lipase activity was not detected, M. restricta has been shown to be deficient in Early log Stationary FFAs Late log Tween-40 3.0 extracellular hydrolases (Mancianti et al., 2000). M. restricta bp std  + φ
bp std  + φ
RT + − + − + − + − + − is fastidious in culture and the lack of detectable lipase 2.0 activity from in vitro cultures does not preclude production in 1.0 LIP1

LIP1 mRNA/ a more natural environment, such as on human skin. ACT1 mRNA ACT1 We have isolated, cloned, and characterized a lipase FFA Tween

ACT1 gene, LIP1, from M. globosa, an organism commonly found Late log Early log Stationary on the scalp and associated with dandruff (Gemmer et al., c d 2002; Gupta et al., 2004a). The conclusion that LIP1 encodes a lipase is based on several arguments. First, a sample of

Subject 1 Subject 5 purified lipase contains a 32 kDa protein with 159-amino-

bp std  + φ
+ − + − bp std  + φ
RT acid residues common to the expected translated product of 2.0 LIP1. Second, the proposed LIP1 translation product shows LIP1 1.0 closest similarity to the lipase 3 family in the Pfam database ACT1 mRNA ACT1 LIP1 mRNA/ and matches many lipases in the GenBank NR database. Third, the proposed LIP1 translation product contains a lipase active site consensus sequence. Fourth, an irreversible lipase Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 ACT1 inhibitor bound to a protein whose sequence matched that predicted for the LIP1 translation product. Fifth, recombinant Figure 6. RT-PCR was used to demonstrate LIP1 expression on human scalp. LIP1 showed lipase activity with the same substrate specifi- (a) In vitro expression under different culture conditions, where early log city as the M. globosa extract. refers to 18 hours, Late log refers to 42 hours, and stationary refers to 66 hours The cloned LIP1 encodes the entire lipase, extending from after inoculation. Tween-40s refers to mDixon medium containing Tween- the sequenced N-terminus to a translation stop signal. 40s but no FFA, and FFAs refers to mDixon medium containing FFA but no s However, it is not clear if the cloned gene encodes the entire Tween-40 . These latter two samples were collected at late log phase translation product. Although there is an upstream methionine (42 hours). DNA size standards are l digested with HindIII and jX digested codon encoded by the cloned gene, it is unclear whether this with HaeIII. (b) Relative expression levels expressed as a ratio of LIP1 to ACT1 mRNA. (c) In vivo expression on human scalp. (d) Relative in vivo expression is a translation-initiating methionine or an internal part of a pro levels expressed as a ratio of LIP1 to ACT1 mRNA. The error bars represent the sequence, commonly found in other lipases, including a standard error for four independent PCR reactions on four cDNAs. similar Rhizopus lipase (Beer et al., 1996). LIP1 appears to be quite different from the recently cloned lipase genes from M. furfur (MfLIP1, Brunke and Hube, 2006) tion of LIP1 expression, with higher LIP1 expression in the and M. pachydermatis (EST1, Shibata et al., 2006). No presence of a more complex lipid source. significant alignment was produced using LIP1 protein sequence search against either MfLIP1 or EST1 sequences LIP1 was expressed on human scalp using default BLASTP (Altschul et al., 1997) parameters, A small set of human subjects was chosen to see if LIP1 whereas MfLIP1 and EST1 show significant similarity to each expression could be detected on a human scalp. mRNA was other (41% identity). Both MfLIP1 and EST1 show homology isolated from the swab samples and served as a template in a to the lipase genes of Candida albicans, whereas LIP1 shows reverse transcriptase reaction followed by a PCR, using two little homology with C. albicans lipase gene families. A Pfam primers designed to amplify the lipase gene. Gel electro- (release 20, Bateman et al., 2004) search indicates MfLIP1 phoresis analysis of the RT–PCR products indicated a nucleic and EST1 belong to the LIP lipase family (secretory lipase, acid band of 397 bp, the size expected for the LIP1 cDNA accession number: PF03583), and LIP1 belongs to lipase 3 PCR product (Figure 6c). A control reaction without reverse family (lipase class 3, accession number: PF01074). The transcriptase produced no such DNA fragment (Figure 6c), lipase class 3 family is called ‘‘class 3’’ because its lipases are indicating that the observed RT–PCR product was not a result not closely related to other lipase families. Taken together, of amplification of contaminating genomic DNA. The LIP1 appears to encode a novel type of Malassezia lipase. RT–PCR product amplified from human scalp was cloned The lipase activity of M. globosa cells hydrolyzed and sequenced, and the cloned sequence matched the monoglyceride, diglyceride, and triglyceride substrates, expected gene fragment (data not shown). LIP1 mRNA was whereas the extracted lipase only hydrolyzed monoglyceride detected on the scalp of three of five subjects (Figure 6c and d). and diglyceride substrates. Some possible explanations for this difference are (1) there is more than one lipase produced DISCUSSION in vitro, (2) the lipase was modified during extraction and lost Lipase activity was produced by cultures of four species of activity against the triglyceride substrate, or (3) the extraction Malassezia. For two of these species, M. furfur and M. removed a cofactor or emulsifying factor necessary for pachydermatis, lipase activities have been described pre- triglyceride hydrolysis. There are precedents for fungi with viously, although with different substrates. This report is the multiple lipase genes. For example, C. albicans encodes at first description of lipase activities from M. globosa and M. least 10 lipases (Hube et al., 2000). There are also precedents

2142 Journal of Investigative Dermatology (2007), Volume 127 YM DeAngelis et al. Malassezia globosa Lipase Gene Cloning and Expression

for the substrate specificity observed for LIP1, as other fungal Skin surface lipases have been identified that are active against diglycerides Malassezia Secretion layer and monoglycerides but not triglycerides (Isobe et al., 1994; Lipids, shed skin cells, Toida et al., 1995; Tsuchiya et al., 1996). However, none of proteins these enzymes is among those lipases showing highest amino- Growth, acid sequence similarity to the M. globosa lipase. proliferation LIP1 was expressed on human scalp, as RT–PCR indicated thepresenceoftheLIP1 mRNA. As far as we know, this is but M.globosa the second example of researchers demonstrating gene cell expression of a fungus on human skin, with the first example Secretion layer set with C. albicans protease genes (De Bernardis et al., 2004). As this technology is applied to a wider range of genes and Corneocyte organisms, it should provide invaluable insight to host–microbe interactions. Future work could include testing for correlations Saturated Lipase between lipase gene expression and D/SD symptoms. fatty acid secretion The majority of recent evidence indicates Malassezia play consumption a key role in dandruff. A variety of antifungal agents (zinc Triglyceride pyrithione, ketoprofen, selenium sulfide, octopirox, and Unsaturated hydrolysis ciclopirox olamine) reduce the number of Malassezia on fatty acid penetration the scalp, correlating closely with a reduction in dandruff symptoms (Schwartz et al., 2004). M. globosa has been found on human scalps (Gemmer et al., 2002). Malassezia can Pathogenicity increase levels of FFAs and lower levels of triglycerides (Ro and Dawson, 2005). In a model of D/SD, lipase-mediated lipid hydrolysis would provide the fatty acids required for M. globosa growth, with excess fatty acids penetrating the Figure 7. A model for D/SD etiology due to Malassezia lipase-mediated stratum corneum and leading to barrier disruption (Figure 7). hydrolysis of scalp lipids. Some fatty acids are consumed by the fungal cells, Further support for this model is evidenced by the dandruff- whereas other fatty acids effect scalp irritation. like flaking induced by application of oleic acid, a by-product of degradation of human sebum, to the scalps of susceptible expression experiment, two modifications were used: (1) Tween- individuals (DeAngelis et al., 2005). 40s was removed, and an additional 1% of oleic acid was added In this paper, we describe the isolation of a M. globosa (labeled ‘‘no Tween-40s, þ FFA’’), and (2) oleic acid was removed, lipase and the corresponding gene and show that the gene is leaving 1% Tween-40s as the sole lipid source (labeled ‘‘Tween- expressed on human scalp. Given the likely importance of 40s, no FFA’’). The cultures were incubated at 311 for 1 week after M. globosa lipid metabolism to D/SD, we have taken the the culture turned opaque, which typically takes 5–7 days. The cells first step toward a molecular description of this process. The were harvested by centrifugation, washed with 100 mM Tris–HCl (pH LIP1-encoded lipase was active against diglycerides but not 8.0), and frozen at À801. The cells were thawed at room temperature triglycerides, suggesting that additional lipases are involved in and suspended in 8 ml of extraction buffer (25 mM Tris–HCl (pH 8), scalp lipid metabolism, as triglycerides are in greater 90 mM NaCl, 8 mM KCl, and 1 mM CaCl2) per gram of cells. abundance than diglycerides in both newly expressed (Stewart Lipase was recovered by incubating the cell suspension for 2 hours et al., 1978) and accumulated sebum (Whestley, 1986; Wertz at room temperature, mixing on a Lab Rotator (Lab-Line, Melrose and Michniak, 2000). It therefore remains likely that additional Park, IL). The cells were spun for 10 minutes at 3200 g, and the lipases are present in Malassezia, and further work will be supernatant collected. The cells were extracted multiple times, with necessary to delineate the complete metabolic pathway. the active extracts combined. The pooled supernatant was centrifuged at 45,000 g for 1 hour, yielding a clarified supernatant. Ammonium MATERIALS AND METHODS sulfate was added to 25% (w/v) saturation, and insoluble material Culture of malassezia and recovery of M. globosa 7966 lipase removed by centrifugation (45,000 g for 1 hour). The sample was All Malassezia strains (Centraalbureau voor Schimmelcultures, dialyzed against buffer A (22 mM 2-morpholinoethanesulfonic acid

Utrecht, The Netherlands) were cultured in mDixon medium (per (MES; pH 5.5), 80 mM NaCl, 8 mM KCl, and 1 mM CaCl2), loaded onto liter water: 36 g malt extract (Difco, Detroit, MI), 20 g desiccated ox a MonoQ anion exchange column (Amersham Biosciences, Piscat- s bile, 10 ml Tween-40 , 6 g peptone (Difco, Detroit, MI), 2 ml away, NJ) with a Pharmacia Akta Explorer 100 FPLC, and eluted with glycerol, 2 ml oleic acid (crude food grade; J.T. Baker, Phillipsburg, a linear gradient of 0–250 mM NaCl in buffer A. Fractions with lipase NJ), pH 6.0 (with 1 N HCl) with 500 mg/ml chloramphenicol. activity were pooled and concentrated. The sample was then run on a Malassezia strains reported here are M. globosa CBS 7966, M. furfur native polyacrylamide gel that was subsequently cut into 5 mm slices. CBS 7982, M. obtusa CBS 7968, M. restricta CBS 7877, M. slooffiae CBS 7956, M. sympodialis 7977, and M. pachydermatis ATCC Lipase assays (Manassas, VA) 74522. Chemicals were obtained from Sigma- In one assay for lipase activity, we used 160 mM p-nitrophenyl oleate Aldrich (St Louis, MO), unless otherwise indicated. For the LIP1 gene as a substrate in buffer A. After a 20-minutes incubation, Tris–HCl

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(pH 8.0) was added to bring the pH to 8, and absorbance measured a Thermo Spectronic (Rochester, NY) French Press at 30,000 psi. The at 410 nm. Total protein was determined using a commercial kit ruptured cell debris was removed by centrifugation. The supernatant (Pierce Chemical Co., Rockford, IL). was twice extracted with phenol:chloroform:isoamyl alcohol Lipase activity was also determined by gas chromatography. (25:24:1) and once with chloroform. The DNA was precipitated Lipase samples, in 250 ml Dulbecco’s phosphate-buffered saline, with 2.5 volumes ethanol, 1/10 volume 3 M sodium acetate (pH 5.2), were mixed with either triolein or a 50:50 mixture of triolein and collected by centrifugation and suspended in 2 ml water. 0 1,2-diolein (125 ml of 1.25 mM lipid in 1 M MES (pH 5.5), 2.5% A degenerate primer (5 -GAYCARCCNGTNGCNAAYCCNTAY 0 isopropanol) with 560 ml1M MES (pH 5.5). After 24 hours of gentle AA-3 , where R ¼ A þ G, Y ¼ C þ TandN¼ A þ G þ C þ T; supplied shaking at 371, solutions were acidified with a drop of concentrated by Sigma-Genosys, The Woodlands, TX) was designed, based on the HCl and centrifuged. The lipid phase was weighed and diluted with amino-acid sequence DQPVANPYN, a portion of the sequence 0 CHCl3 containing tricaprin and tridecanoic acid as internal determined from the N-terminus of the lipase. Another primer (5 - standards. A portion was dried and mixed with derivatizing reagent CCRTGNACRTTYTCYTGNCCNGGRTA-30)wasbasedonthecom- (N-trimethylsilylimidazole and bis(trimethylsilyl)triflouroacetamide plement of a strand encoding amino-acid sequence YPGQENVHG. (both from Pierce Chemical Co., Rockford, IL)) and pyridine. After M. globosa genomic DNA (2 ml) was used as a template for PCR 1 hour at 901C, the samples were analyzed using a Hewlett-Packard with 1 mg of each primer, with Amplitaq DNA Polymerase (Applied 5890 Series II gas chromatography and a fused silica capillary Biosystems) according to the manufacturer’s specifications. The PCR column coated with a 0.12 mm film of CP-Sil 2 CB stationary phase was performed in a PTC-200 Peltier Thermal Cycler (MJ Research, (Varian Inc., Palo Alto, CA). Carrier gas was helium at B1 ml/minute Watertown, MA) beginning with a 941 treatment for 3 minutes flow rate. Injector and detector temperatures were 3501C and the followed by 25 cycles of 1 minutes at 941, 2 minutes at 551, and column temperature was programmed to hold at 801C for 2 minutes, 3 minutes at 721. PCR (50 ml) reaction was sent to Seqwright ramped to 3501Cat201C/minute and held for 2 minutes. Analyte (Houston, TX) for gel purification of the 700 bp product and primer peak areas were normalized against the appropriate internal extension sequencing. standard and adjusted for sample weight before comparison to the For RACE (Frohman et al., 1988), a SMART RACE cDNA control incubations without enzyme. Amplification Kit (Clontech, Palo Alto, CA) was used to generate Lipase activity was also measured using a glycerol detection assay RACE-ready M. globosa cDNA. A primer (50-ACAGCACGAGCGC (McGowan et al., 1983). The samples were assayed in buffer A for GAAGC-30), was used in the rapid amplification of 30 cDNA ends 1 hour with vigorous shaking and the pH adjusted with 50 mlof1M according to the manufacturer’s specifications. Gel electrophoresis Tris–HCl (pH 8.0). Glycerol detection reagents (Molecular Probes, of the completed RACE reaction revealed a DNA fragment of Eugene, OR) were added. The samples were shaken for 15 minutes at approximately 400 bp. The product was purified using a Qiaquick

room temperature, and OD540 was measured. For each sample, there PCR purification kit (Qiagen, Valencia, CA) and ligated to the vector was a control with the lipase boiled for 10 minutes. These controls pCR 2.1-TOPO using the TOPO TA cloning kit (Invitrogen). The

always produced an OD540 of o0.15. When other pH conditions insert was sequenced. were tested, we (1) substituted Tris–HCl (pH 8.0) for MES (pH 5.5) and A primer (50-CATTTTCCGTGCTGTCACAGTA-30) was used in the (2) replaced the MES and salts with phosphate-buffered saline. amplification of 50 cDNA ends, with a B160 bp product on an agarose gel. The RACE reaction product was purified, cloned, and Peptide sequencing sequenced. To generate a single fragment spanning the lipase gene Partially purified lipase was electrophoresed under denaturing segments identified in separate PCR reactions, two oligonucleotides conditions on a 10–20% mini-gel (Invitrogen, Carlsbad, CA). The (50-ATGCTCTTCAGTCGCTTT-30;50-TTAATGAGCACCAACCTG-30) gel was electroblotted to a polyvinylidene difluoride membrane were used for amplification of genomic DNA. The resulting PCR (Fluorotrans, Pall Life Sciences, East Hills, NY) and stained with product was cloned and sequenced. Coomassie blue. The band migrating at B32 kDa was excised from the blot for N-terminal sequencing on a Procise 494 HT protein Production of recombinant LIP1 sequencer (Applied Biosystems, Foster City, CA). LIP1, encoding Ser20 through His304, was expressed in Pichia To obtain the sequence of individual peptides encoded by LIP1, pastoris using pPICZ (Invitrogen). The secreted lipase was isolated the B32 kDa gel electrophoresis band was excised and digested using Source30Q cation exchange, octyl sepharose, and Source 15S with trypsin (Keough et al., 2000). The tryptic peptides were chromatography (all resins from GE Healthcare, Piscataway, NJ). separated by reverse-phase HPLC using a Waters Delta-Pak

2 Â 150 mm C18 column (5 mm particles, 300A˚ pore size, Waters Synthesis of covalent lipase inhibitor Corp., Milford, MA), applied to a precycled glass fiber filter, and The biotinylated suicide inhibitor (Figure 5a) was synthesized using a sequenced on the Procise sequencer. Mass spectrometry-based scheme similar to that of Deussen et al. (2000). Biotinylated poly- sequencing employed sulfonic acid derivatization followed by oxoethylene amine was synthesized using acid chloride activation of matrix-assisted laser desorption ionization post source decay or biotin and subsequent reaction with 4,7,10-trioxa-1,13-tridecane- electrospray ionization tandem mass spectrometry methods as diamine. The 4-nitrophenyl-activated phosphonate with the oxo- described (Keough et al., 1999; Keough et al., 2002). undecyl tether was activated as the N-hydroxysuccinimide ester and subsequently reacted in dimethylformamide with the biotinylated Molecular biology poly-oxoethylene amine to yield the inhibitor. Purification consisted To prepare genomic DNA, 5 g of washed M. globosa cell pellet was of flash chromatography (silica gel, 5–11% methanol in methylene 1 suspended in 37 ml 0.2 N NaOH, 1% SDS, and passed twice through chloride) to yield the inhibitor as a colorless oil. H NMR and

2144 Journal of Investigative Dermatology (2007), Volume 127 YM DeAngelis et al. Malassezia globosa Lipase Gene Cloning and Expression

electrospray mass spectrometry were used to verify compound as Amount and quality of cDNA was normalized by PCR using actin having the proposed structure. gene-specific primers (50-GGTTACCCATTCACGACGAC-30,50-GCAA 1 0 H NMR (500 MHz, CDCl3) d 1.24–1.83 (m, 31H), 1.94 (m, 2H), 2.21 GAATCGAACCACCAAT-3 ). Actin cycling protocol was 1 Â (5 minutes (t, 2H, J ¼ 7.3 Hz), 2.75 (d, 1H, J ¼ 12.9 Hz), 2.92 (dd, 1H, J ¼ 12.9 Hz, at 941C, 1 minute at 601C, 1 minute at 721C), 23 Â (1 minute at 941C, J ¼ 4.9 Hz), 3.16 (m, 1H), 3.27 (m, 2 H), 3.36 (m, 2H), 3.53–3.7 (m, 12H), 1 minute at 601C, 1 minute at 721C), 1 Â (5 minutes at 941C, 1 minute at 4.02 (t, 2H, J ¼ 6.6 Hz), 4.12–4.29 (m, 2H), 4.34 (m, 1H), 4.52 (m, 1H), 601C, 10 minutes at 721C). LIP1-specific amplifications were similarly 5.95 (s (br), 1H), 6.56 (s (br), 1H), 7.39 (d, 2H, J ¼ 9 Hz), 8.24 (d, 2H, performed except for the use of 30 cycles. LIP1-specific primers had the J ¼ 9 Hz) MS (electrospray) m/z 874.4 (M þ H), 896.4 (M þ Na). sequence: forward 50-GACAGCACGGAAAATGGTCT-30; reverse 50-CA TCCATACGCAGCTCAATG-30. The PCR-generated product from both in Isolation of Inhibited Lipase vitro Malassezia cultures and in vivo scalp samples were cloned and An M. globosa culture (40 ml) was pelleted, washed, and suspended sequenced to confirm they were the correct product (data not shown). s in buffer B (50 mM Tris–HCl (pH 8.0), 100 mM NaCl), and divided into The amount of PCR product was determined using SybrGreen two samples of 3.3 ml each. Inhibitor (5 ml, Figure 6a, 25 mM stock in (Molecular Probes) with fluorescent densitometry of digitally captured DMSO) was added to one sample, and DMSO (5 ml) was added to gel images (Kodak EDS 290, Rochester, NY). the other sample. The reaction was mixed by inversion overnight in the dark at room temperature. CONFLICT OF INTEREST The reaction mix was pelleted, washed three times with 1 ml of The authors state no conflict of interest. buffer B, and suspended in 150 mlof2Â Tris-Glycine SDS gel- loading buffer (Invitrogen). After boiling and a spin, the supernatant ACKNOWLEDGMENTS was transferred to a tube with 12 ml of buffer B. We thank Melissa Monich, Joe Listro, Jim Dunbar, Pete Ellingson, Chris Kelling, Bob Lindenschmidt, Bob Leboeuf, John McIver, and Charlie Bascom Avidin-coated beads (220 ml, Sigma, part A-9207, with a loading for their support and Blue Sky Biotech, Inc. for the production of recombinant of 3.8 nM/20 ml beads) were washed three times with 1 ml of buffer B LIP1. The Genebank accession number of LIP1 is DD210404. plus 0.1% SDS and suspended in 1 ml of buffer B. Bead suspension SUPPLEMENTARY MATERIAL (200 ml) was added to each reaction. The samples were mixed by Figure S1. Sequence of the lipase gene and predicted translation product. inversion for 25 hours. The beads were collected by centrifugation, Figure S2. Multiple sequence alignment (Clustal W (1.83), Higgins et al., washed three times with 1 ml of buffer B plus 0.1% SDS and with 1994) with the top four hits of L1P1 in nr database. 1mlof50mM Tris–HCL (pH 8.0). The beads were treated with one-eighth concentrated gel-loading REFERENCES buffer, boiled, and spun. The process was repeated. The supernatants Altschul S, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W et al. (1997) were combined, concentrated by drying, and analyzed by SDS Gapped BLAST and PSI-BLAST: a new generation of protein database polyacrylamide gel electrophoresis. search programs. Nucleic Acids Res 25:3389–402 Ashbee HR, Evans EGV (2002) Immunology of diseases associated with RT-PCR Malassezia species. 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