Isolation and Expression of a Malassezia globosa Lipase Gene, LIP1 Yvonne M. DeAngelis, Charles W. Saunders, Kevin R. Johnstone, Nancy L. Reeder, Christal G. Coleman, Joseph R. Kaczvinsky, Celeste Gale, Richard Walter, Marlene Mekel, Martin P. Lacey, Thomas W. Keough, Angela Fieno, Raymond A. Grant, Bill Begley, Yiping Sun, Gary Fuentes, R. Scott Youngquist, Jun Xu, Thomas L. Dawson  Journal of Investigative Dermatology  Volume 127, Issue 9, Pages 2138-2146 (September 2007) DOI: 10.1038/sj.jid.5700844 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Lipase activity was detected in four different Malassezia species. Values represent pmoles of p-nitrophenol released per microgram total cell protein per minute incubation. The reaction was carried out as described in the Materials and Methods section except that that the incubation was for 15minutes at 30° and 1.3% Triton X-100 was included in the assay buffer. Error bars represent standard error of three replicate experiments. Malassezia strains reported here are M. globosa CBS 7966, M. furfur CBS 7982, M. obtusa CBS 7968, M. restricta CBS 7877, M. slooffiae CBS 7956, M. sympodialis 7977, and M. pachydermatis ATCC 74522. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Gas chromatography analysis reveals that isolated lipase is active against diolein and not triolein. Extracted and cell-based lipase activity were compared, using (a) a triolein/diolein mixture and (b) triolein as a substrate, with the values, on a linear scale, indicating the amount of oleic acid (OA), monoolein (MO), diolein (DO), and triolein (TO) present after incubation. The composition of the substrate alone is indicated in the control samples at the left-hand side of the figure. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 LIP1 hydrolyzed diolein and monolein but not triolein. Values 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 subtracted from the OD540 of the lipase test sample, with the difference in OD540 recorded on the graph. Error bars indicate the standard deviations of the background-subtracted values, with 12 replicates of the samples containing extract and cells and four replicates of the samples containing recombinant LIP1. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Lipase was isolated using native polyacrylamide gel electrophoresis. Native gel fractions were assayed for the presence of lipase, using p-nitrophenol oleate. Fractions 8 and 9 of the native gel were pooled and analyzed by SDS polyacrylamide gel electrophoresis. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

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. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 RT-PCR was used to demonstrate LIP1 expression on human scalp. (a) In vitro expression under different culture conditions, where early log refers to 18hours, Late log refers to 42hours, and stationary refers to 66hours after inoculation. Tween-40® refers to mDixon medium containing Tween-40® but no FFA, and FFAs refers to mDixon medium containing FFA but no Tween-40®. These latter two samples were collected at late log phase (42hours). DNA size standards are λ digested with HindIII and ϕX digested 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 levels expressed as a ratio of LIP1 to ACT1 mRNA. The error bars represent the standard error for four independent PCR reactions on four cDNAs. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 A model for D/SD etiology due to Malassezia lipase-mediated hydrolysis of scalp lipids. Some fatty acids are consumed by the fungal cells, whereas other fatty acids effect scalp irritation. Journal of Investigative Dermatology 2007 127, 2138-2146DOI: (10.1038/sj.jid.5700844) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions