Biochimica et Biophysics Acra 959 (1988) 153-159 Elsevier

153

BBA 52761

Molecular cloning of a phospholipid-cholesterol acyltransferase

from Aeromonus hydrophifa Sequence homologies with lecithin-cholesterol

acyltransferase and other lipases

Julian Thornton a, S. Peter Howard b and J. Thomas Buckley a a Department of Biochemistry and Microbiology, University of Victoria, Victoria (Canada)

and b Centre de Biochimie et de Biologie Moleculaire, CNRS, Marseille (France)

(Received 13 August 1987)

Key words: Acyltransferase; Glycerophosphohpid-cholesterol acyltransferase; Phospholipid-cholesterol

acyltransferase; Phosphatidylcholine-sterol acyltransferase; Nucleotide sequence; Gene clone;

Sequence homology; (A. hydrophila)

We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophircl phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransfera~. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supematant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.

Introduction

The glycerophospholipid-cholesterol acyltrans- ferase (GCAT) released by members of the K’ibrio family shares a number of properties with the mammalian enzyme lecithin-cholesterol acyltrans- ferase (LCAT, [l-4]). Like the plasma enzyme, GCAT will catalyze fatty acid transfer between

phosphatidylcholine and cholesterol, and acyl transfer is 2-position specific. Both enzymes will act as phospholipases in the absence of acyl accep- tors, and neither requires calcium. The microbial enzyme has much less stringent requirements for the acyl donor than does LCAT, as all the com-

Abbreviations: GCAT, glycerophosphohpid-cholesterol acyl-

transferase; LCAT, lecithin-cholesterol acyltransferase (EC

2.3.1.43).

Correspondence: J.T. Buckley, Department of Biochemistry and Microbiology, University of Victoria, Victoria BC, Canada

V8W 2Y2.

mon glycerophospholipids will function as sub- strates, and under some conditions GCAT will hydrolyse other hydrophobic substrates such as cholesteryl ester. As with LCAT, the selectivity for the acyl acceptor is considerable and appears to depend upon specific interactions between the phospholipid and cholesterol.

The primary structure of human LCAT has recently been deduced from the nucleotide se- quence of the cDNA [5] and confirmed by protein sequencing [6]. Two regions of the molecule have been identified which are homologous with amino acid sequences in porcine pancreatic lipase and bovine milk lipoprotein lipase [7]. Each contains a serine. Based on the experimental evidence ob-

tained with porcine lipase, one site, found in a variety of lipases [8], is believed to be involved in substrate binding. The other is thought to be the active site. In spite of these observations, nothing is known of the role of individual amino acids in the reactions catalysed in the lipases, nor is it

0005-2760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

154

known what portions of the molecule participate in the unusual reaction mechansm of LCAT. None of the enzymes so far compared to LCAT prefer- entially catalyses acyl transfer. For these reasons we felt it especially important to determine the sequence of the microbial acyltransferase. In this communication we show that sequences similar to the binding and active sites of the mammalian lipases can be identified in GCAT.

Experimental procedures

Materials Restriction enzymes and DNA modification

enzymes were purchased from Pharmacia or Boehringer. Replicative form DNA of Ml3 mp18 and mp19 and Ml3 17-base primer were obtained from Pharmacia and New England Biolabs. Deoxy and dideoxynucleotide triphosphates were from Pharmacia. [a- 32P]dATP was purchased from Amersham. Other chemicals were the purest com- mercially available.

Bacterial strains, plasmids and media

The bacterial strains and plasmids used in this study and their sources are listed in Table I. The organisms were grown in YT medium, supple- mented where specified with antibiotics at the following concentrations: ampicillin, 200 pg/ml; kanamycin, 50 pg/ml. Blood agar was prepared by adding 20 ml of human blood to 500 ml of tryptic soy agar tempered to 50 o C.

TABLE I

BACTERIA, PLASMIDS AND BACTERIOPHAGES

Strain

E. coli

Genotype or description Source

recA13, hsdS20, aral4, proA E.E. Ishiguro

lacY1, gaIK2, IeuB6, rpsL20

~~115, mtll, supE44

E. co/i JM105 pro-lac, thi, rpsL, hsdR4, Pharmacia endA, sbcB, F’traD36,

proA+ B+, lacIq, 1acZ Ml5

pVK102 KmR, TcR E.W. Nester pBR322 ApR, TcR E.E. Ishiguro pHEc1 See text S.P. Howard PHEc2.2 See text This study mp18/mp19 Ml3 sequencing vectors Pharmacia

DNA preparation A. hydrophila Ah65 chromosomal DNA was

isolated by the method of Puhler and Timmis [9]. The DNA was partially digested with Sal1 (0.3 U SalI/pg chromosomal DNA) for 1 h at 37 o C and fragments of approx. 25 kb were isolated by agarose electrophoresis and further purified by centrifugation through a lo-40% sucrose density gradient as described by Maniatis et al. [lo]. Plasmid DNA, when used for cloning or restric- tion mapping, was isolated via the cleared lysate method of Godson and Vapnek [ll] and purified by centrifugation in a cesium chloride-ethidium bromide gradient as described by Maniatis et al. [lo]. Minipreparations of plasmids, used for screening of insert size in cloning experiments were isolated according to the method of Birn- boim and Doly [12].

Construction of the pVKlO2 gene bank and detec- tion of clones containing GCA T-expressing plasmids

The wide host range cosmid pVK102 [13] was digested with Sal1 and dephosphorylated with calf intestinal alkaline phosphatase. The digested cosmid was mixed with the size-selected Sal1 par- tial digest of A. hydrophifa chromosomal DNA and ligated overnight at 12°C. Aliquots contain- ing 1.2 /.tg of the mixture were packaged in vitro into lambda phage particles using a commercial packaging kit (Amersham) and used to transduce E. coli HBlOl. Detection of clones containing GCAT-expressing plasmids was based on our ob- servation that cells which produce the enzyme (including Aeromonas salmonicida and aerolysin negative mutants of A. hydrophila), exhibit alpha hemolytic phenotypes after 48 h when plated on human blood agar. Positive clones detected in this way were tested for acyltransferase activity as described by MacIntyre and Buckley [l].

Subcloning metho& Vector DNA was pBR322, or the RF DNA of

Ml3 mp18 or mp19. Target DNA was the recom- binant plasmid pHEC1 which contains a 18 kb insert of A. hydrophila with the entire GCAT gene. Subcloning procedures were carried out es- sentially as described by Maniatis et al. [lo].

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DNA sequence analysis The plasmid pHEC2.2, which contains a 1.2 kb insert in pBR322, was digested with the restriction enzymes shown in Fig. 1, either alone or in combi- nation, and the resulting fragments were inserted into the polylinker cloning regions of Ml3 mp18 and mp19 [14]. The DNA inserts were sequenced using the dideoxy chain termination method of Sanger et al. [15] according to the strategy de- picted in Fig. 1. The universal 17-base single stranded primer was used to begin sequencing from the 3’ end of the inserts in each of the recombinant mp18 and mp19 bacteriophages. Where indicated in Fig. 1, sequencing was con- tinued using 18-base oligonucleotide primers which were synthesized using a Sam One DNA syn- thesizer (Biosearch Inc.).

Cell fractionation procedures Shock fluids were obtained by the sucrose-

EDTA method of Willis et al. [16]. The osmoti- cally shocked cells were disrupted by passing them through a French pressure cell (1100 kg/cm*). RNAase, a periplasmic marker, was assayed by measuring the release of acid-soluble adenylate from [ ‘Hlpolyadenylate [17]. The intracellular marker glutamate dehydrogenase was assayed as described by Halpem and Lupo [18]. Enzyme activities for all fractions are given in units/ml of original culture.

Results and Discussion

Isolation of GCA T-containing clones Acyltransferase activity was detected in dis-

rupted cells after overnight growth of one of the alpha hemolytic clones. This confirmed the pres- ence of the gene for GCAT rather than expression of a modified aerolysin gene or a clone of another phospholipase which could presumably also have been selected by the screening procedure. A large amount of the cosmid, containing an 18 kb insert, was purified from a cleared lysate by cesium chlo- ride ccntrifugation. This cosmid (pHEC1) was re- stricted with a variety of endonucleases and the resulting fragments were ligated to the ap- propriately restricted subcloning vector pBR322. One of the subclones, identified on the basis of alpha hemolytic phenotype as well as enzyme pro-

Fig. 1. Strategy for sequencing the GCAT gene. Arrows repre- sent sequences obtained using fragments produced by restric- tion enzyme digestion at the sites indicated or sequences

obtained using synthetic oligonucleotides as primers.

duction, is depicted in Fig. 1. The recombinant plasmid pHEC2.2 contains a 1.2 Kb Pstl frag- ment inserted into the PstI site of pBR322.

Location of GCA T in E. coli clones Most of the acyltransferase activity was re-

covered in the shock fluid of cells containing pHEC1 and pHEC2.2 (Table II). No enzyme was detected in the culture supernatant, indicating that GCAT is not released by E. coli. Similar observa- tions have been made with a variety of extracellu- lar proteins cloned into this species, leading to the conclusion that E. coli lacks a mechanism for the export of proteins across its outer membrane [19].

Nucleotide sequence of the GCAT gene Using the sequencing strategy shown in Fig. 1,

the nucleotide sequence containing the GCAT gene was determined. The results are presented in Fig. 2. The nucleotide sequence corresponding to the beginning of the protein was identified by compar- ing the translated amino acid sequence with the previously determined amino terminal sequence of GCAT purified from A. salmonicida. Only one of the first 18 amino acids is different in the enzymes from the two species. GCAT from A. salmonicida contains a threonine at position 3 rather than a serine (data not shown here). The open reading frame shown in the figure encodes a protein of 281 amino acids with a molecular weight of 31303. This is considerably larger than the molecular weight of the protein from A. salmonicida as determined by sodium dodecyl sulphate electro- phoresis [l]. This may reflect major differences between the two proteins. (In spite of the similar- ity of their amino termini, plyclonal antibody pre-

156

CCGACAcrccorax;ca:~rn~cx:crcAa:ncnccATCAATCAGCCATPCC~TCA(SPA 1266 I.278 1290 1302 1314

P&I ECWl'CATpGATXQITCIGa3;cATGGl!GGcCAGocXCICCECAG

1332 1344 1356 1368

Fig. 2. DNA sequence of the GCAT gene. The nucleotide residues are numbered in the 5’ to 3’ direction. The coding region is translated above the DNA sequence. Numbers below the sequence refer to the nucleotide positions, numbers above to the amino acid

positions. The sequence from - 18 to - 1 is the probable signal sequence. The sequence corresponding to the ammo acid sequence of

the enzyme from A. salmonicida is underlined (see text). Several restriction sites are identified for reference to Fig. 1.

pared against A. salmonicida GCAT did not cross-react with A. hydrophila GCAT.) Alterna- tively, GCAT may be processed post-transla- tionally. We have shown that another protein pro- duced by this species, the hole-forming toxin aerolysin, is released from the bacteria as a proto- xin which is activated by removal of approxi- mately 20 amino acids from the carboxy terminus

WI. The codon usage frequency in the translated

reading frame is very similar to the frequency we have observed for the aerolysin gene of this species (not shown here). In contrast to the aerolysin gene [21], there are no obvious promoter and termina- tor regions surrounding the gene for GCAT, which would suggest that it is translated from a poly- cistronic message.

Comparison with the amino acid sequences of other lipases

A search of the National Biomedical Research Foundation protein sequence library [22] failed to reveal any sequences with extended regions ho- mologous to GCAT. Recently, however, several authors have reported sequence similarities among

157

a variety of lipases [7,8,23]. The results in Fig. 3A show that GCAT contains a sequence which is highly conserved in lipases with different origins and different biochemical properties. Based on data obtained with porcine pancreatic lipase [24], this region is believed to form the interfacial lipid-binding site of these enzymes. Komaromy and Schotz [8] have noted that hepatic lipase contains two such sequences and they suggest that this enzyme may be able to bind lipids more efficiently than the other enzymes, thereby eliminating the need for protein cofactors. GCAT contains only a single copy of the binding se- quence, however, and it has no cofactor require- ments. GCAT also contains a sequence similar to regions in several lipases which Maraganore and Heinrickson [7] suggest contain the active sites of these enzymes. The data in Fig. 3B indicate that, although the apparent homology is not convicing, this region in GCAT is if any thing more similar to pancreatic lipase than is the sequence in LCAT. Interestingly, like pancreatic lipase, GCAT is in- sensitive to diisopropylphosphofluoroidate at con- centrations which completely inhibit LCAT [25]. Like the other lipases, GCAT is a hydrophilic

TABLE II

DISTRIBUTION OF ACYLTRANSFERASE AND MARKER ENZYMES AFTER OSMOTIC SHOCK

Shocked cells were French pressed after shocking to release their contents.

HBlOl

supematant

wash

shock fluid

shocked cells

HBlOl (pHEC1)

supematant

wash shock fluid

shocked cells

HBlOl (pHEC2.2) supematant

wash

shock fluid

shocked cells

Acyltransferase Glutamate

(nmol cholesteryl ester ( p mol NADH oxidized

formed/min per ml) /min per ml)

U/ml (W) U/ml (W)

0 0

0 0

0 0

0 0.48 (100)

0 (0) 0

0 (0) 0 5.5 (75) 0

1.8 (25) 0.36 (100)

0.1 (1.4) 0

0 0

7.0 (89) 0.06 (15)

0.8 (9.7) 0.32 (85)

RNAase

(nmol adenylate released

/mitt per ml)

U/ml (%)

0

0.18 (12)

0.94 (65)

0.34 (23)

0

0.12 (7) 1.22 (75)

0.27 (8)

0

0.12 (8)

0.80 (61)

0.42 (31)

158

A: Lipid Binding Domain

MicrcbialGCAT

Rat heptic lips2

~lippmteinlipse

Rat lingual lipase

HLmlanLcAT

Fmxine pancreatic 1ipas.e

B:Active site

gAq.Ile.Val.Met.Fbe.Gly.Asp.Ser.Le".Ser

26ker.Val.His.Leu.Phe.Ile.Asp.Ser.leu.Gln

244Ser.Ile.His.Leu.me.Ile.Asp.Ser.IEu.Le"

163Lys.Ile.His.~.Val.Gly.His Ser.Gln.Gly

173F'ro.Val.Rze.I_eu.Ile.Gly.His.Ser.Leu.Gly

145Asn.Val.His.Val.Ile.Gly.His.Ser.Leu.Gly

GCAT 232Trp.Lys.F%-o.Ehe.Ala.Ser.Aq.Sor.Ala.Ser.~.Asp. .Ser.Gln

Pancreatic l%rp.Lys. .Gly.Gly.Ser.Ary.l%r.Gly.Tyr.Thr.Glu.Aa.Ser.Gln lipas

LCAT %l-rp. .Gly.Gly.Ser.Ile.Lys.F?m.Met.Leu.Val.Leu.Ala.Ser.

Fig. 3. Amino acid homology between GCAT and other hpases. A: homologies with the lipid-binding domain of porcine pancratic

lipase and other hpases. The numbers refer to amino acid positions. B: comparison with the proposed active site of porcine lipase and a similar region of LCAT. See text for details.

protein. Analysis of the predicted amino acid se- quence [26,27] indicated an average (H) 19 hy-

dropathy of -0.56 and no long hydrophobic

stretches. Site-directed mutagenesis should establish

whether or not the regions identified in Fig. 3 are actually involved in the GCAT reaction and de- tailed analysis of the sequences of the mammalian and microbial acyltransferases may lead to an understanding of why their reaction mechanisms appear so similar.

Acknowledgement

We gratefully acknowledge the assistance of Margaret Green. This research was supported by the National Science and Engineering Research Council of Canada and the British Columbia Heart Foundation.

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