Purification and characterization of lysophospholipase-transacylase (h-LPTA) from a highly virulent strain of Candida albicans

Mirbod, Fariba; Banno, Yoshiko; Ghannoum, Mahmoud A.; Ibrahim, Ashraf S.; Nakashima, Shigeru; Kitajima, Yasuo; Cole, Garry T.; Nozawa, Yoshinori

doi:10.1016/0005-2760(95)00072-k

Cited by 57 publications

(69 citation statements)

References 30 publications

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“…Reduction in LPL/LPTA activities by sulphydryl blocking (oxidising) agents such as N-ethylmaleimide and Ellman's reagent has been observed in purified preparations from Candida albicans [27] and in the present study, suggesting that cysteine residues are necessary for these activities. The sulphydryl reducing agent, dithiothreitol, inhibited PLB and LPL, but not LPTA from cryptococci, suggesting that these activities are associated with different active sites or separate proteins.…”

Section: Discussionsupporting

confidence: 72%

“…Long-chain acyl carnitines are competitive inhibitors of LPL/LPTA activity in rabbit myocardium [19] and inhibit C. albicans LPTA as well as adherence of the fungus to lysophospholipids and HEp-2 cells [28]. However, palmitoyl carnitine enhanced LPL activity in C. albicans [22,27]. In contrast to the effects of palmitoyl carnitine, carnitine marginally inhibited cryptococcal LPL and LPTA activities, but did not inhibit PLB; therefore the addition of an acyl chain is required for binding to and inhibiting the cryptococcal phospholipases.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Santangelo

Nouri-Sorkhabi

Sorrell

et al. 1999

Journal of Medical Microbiology

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A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31 P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40OC. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption experiments indicated that > 85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37°C could contribute to membrane degradation and virulence.

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Santangelo

Nouri-Sorkhabi

Sorrell

et al. 1999

Journal of Medical Microbiology

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“…The optimum pH of PLB 684 is 8.4 and resembles the optimal pH activity of TAP [10] and LipC [11] but differs from fungal PLBs [6,7,9,[15][16][17], which function optimally under acidic pH conditions (pH 2.5-6). The observed optimum temperature of PLB 684 activity (approximately 50°C) was higher than that of lysophospholipase of Mycobacterium leprae (approximately 37°C) [18].…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, lysophospholipase-transacylase activity is associated with some PLB enzymes, allowing these enzymes to transfer a FFA to a lysophospholipid and produce a diacylphospholipid. Hydrolase and acyltransferase activities have been detected in several fungi, including Saccharomyces cerevisiae [5], Candida albicans [6], Candida utilis [7], Penicillium chrysogenum [8] and Cryptococcus neoformans [9]. PLBs from Escherichia coli [10] and Bacillus subtilis [11] are known; however, there has been no investigation of bacterial PLBs with respect to whether they produce lysophospholipids during diacylglycerophospholipid hydrolysis and possess lysophospholipase-transacylase activity.…”

Section: Introductionmentioning

confidence: 99%

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

et al. 2013

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A novel metal ion-independent phospholipase B (PLB 684 ) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 UÁmg protein À1 ). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50°C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB 684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K m , V max and k cat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mM, 15.8 mmolÁmin À1 Ámg protein À1 and 1.02 9 10 4 s À1 , respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1 : sn-2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB 684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB 684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB 684 and that the active site may include residues Ser330 and His332.

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“…Of the medically important fungi, extracellular phospholipase activities have been described in Candida albicans and Aspergillus fumigatus, but characterized only in the former [17,18]. In C. albicans, LPL, LPTA and PLB activities were identified in crude culture filtrates of the organism, but only enzymes with LPTA activity were purified [18][19][20] ; no PLB activity was detected in purified enzyme preparations [18,21].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

Chen

Wright

GOLDING³

et al. 2000

Biochemical Journal

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Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase\transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH % ) # SO % fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities copurified as a single protein with an apparent molecular mass of 70-90 kDa by SDS\PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing Nlinked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V max values for PLB and LPL activities were 12.3 and 870 µmol\min per mg of protein respectively ; the corresponding

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Purification and characterization of lysophospholipase-transacylase (h-LPTA) from a highly virulent strain of Candida albicans

Cited by 57 publications

References 30 publications

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

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