JOURNAL OF Vol. 269, No. 12, Issue of 25, pp. 9388-9391 ...THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Soeiety for Biochemistry and Mo~ecular Biology, Inc. Vol. 269,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Soeiety for Biochemistry and Mo~ecular Biology, Inc.

Vol. 269, No. 12, Issue of March 25, pp. 9388-9391, 1994 Printed in U.S.A.

Complete cDNA Encoding Human Phospholipid Transfer Protein from Human Endothelial Cells*

(Received for publication, November 17, 1993, and in revised form, January 10, 1994)

Joseph R. Day, John J. AlbersS, Catherine E. Lofton-Day$, Teresa L. Gilbert$, Andrew F. T. Chingg, Francis J. Grant$, Patrick J. O’Hara$, Santica M. Marcovina, and Janet L. Adolphson From the Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, Seattle, Washington 98103 and SZymoGenetics Corporation, Seattle, Washington 98105

Phospholipid transfer protein, with an apparent mo- lecular mass of 81 kDa, was purified from human plasma. The NHz-terminal amino acid sequence of a 51- kDa proteolytic fragment obtained from phospholipid transfer protein allowed degenerate primers to be de- signed for polymerase chain reaction and the eventual isolation of a full-length cDNAfrom a human endothelial cDNA library. The cDNA is 1,750 base pairs in length and contains an open reading frame of 1,518 nucleotides en- coding a leader of 17 amino acids and a mature protein of 476 residues. Northern blot analysis shows a single mRNA transcript of approximately 1.8 kilobases with a wide tissue distribution. The gene was mapped to chro- mosome 20 using a humadrodent somatic cell hybrid mapping panel. Phospholipid transfer protein was found to be homologous to human cholesteryl ester transfer protein, human lipopolysaccharide-binding protein, and human neutrophil bactericidal permeabil- ity increasing protein (20, 24, and 26% identity, respec- tively).

Although both high density lipoprotein (HDL)l levels and particle size have been shown to be inversely correlated with the risk of coronary heart disease (1, 2), the mechanisms un- derlying these associations are not well understood. Evidence thus far suggests that lipid transfer proteins play a key role in regulation of plasma HDL level, size, composition, and inter- conversions. Human plasma contains at least two different lipid transfer proteins: cholesteryl ester transfer protein (CETP) (3,4), also referred to as lipid transfer protein I (5) , and phospholipid transfer protein (PLTP), also referred to as lipid transfer protein I1 (5 ,6 ) . In addition to cholesteryl ester, CETP facilitates the transfer of triglycerides and phospholipids be- tween lipoprotein particles (5), whereas PLTP primarily pro- motes the exchange and transfer of phospholipids (6) . Recently, purified PLTP was shown to promote the conversion of HDL into populations of larger and smaller particles in the absence of other lipoproteins (7).

Program Project Grant HL 30086 (to J. J. A.). The costs of publication * This research was supported by the National Institutes of Health

of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in ac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s1 reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number($ L26232. $To whom correspondence should be addressed: Northwest Lipid

Research Laboratories, 2121 N. 35th St., Seattle, WA 98103. Tel.: 206-

The abbreviations used are: HDL, high density lipoprotein; CETP,

HUVE, human umbilical vein endothelial; PCR, polymerase chain re- cholesteryl ester transfer protein; PLTP, phospholipid transfer protein;

action; LBP, lipopolysaccharide-binding protein; BPI, bactericidal per- meability-increasing protein.

685-3330; Fax: 206-685-3279.

We report, for the first time, the protein and nucleic acid sequences of human PLTP cloned from a human umbilical vein endothelial (HUVE) cell cDNA library.

EXPERIMENTAL PROCEDURES

PLTP was purified from 2 liters of fresh human plasma by modifica- tion of a procedure reported previously (6). After ultracentrifugation of plasma at d 1.21-1.25 g/ml, the resulting fraction was subjected to sequential chromatography by phenyl-Sepharose and heparin-Sepha- rose followed by a Q-Sepharose Fast Flow column, which was run using conditions similar to those reported for DEAE-Sepharose (6). The last two steps of the purification procedure were carried out by butyl-Toyo- pearl 650 and Toyopearl HW-55 chromatography as described for the isolation of CETP (8). Phospholipid transfer activity of purified PLTP was assessed by the transfer of [3Hlphosphatidylcholine from phospho- lipid vesicles to HDL (9). Purified active PLTP was concentrated by lyophilization, subjected to 1&20% SDS-polyacrylamide gradient gel electrophoresis, and electroblotted onto a polyvinylidene difluoride membrane. The two bands (the mature 81-kDa protein and a 51-kDa proteolytic fragment of PLTP) were excised and their NH,-terminal amino acid sequences determined with an Applied Biosystems model 470A Sequencer (Foster City, CA) using the manufacturer’s program- ming and chemicals. An antihuman PLTP antibody was obtained by immunizing a New Zealand White rabbit with two intramuscular in- jections of 50 pg of the 51-kDa fragment of PLTP.

cDNA libraries were constructed from HepG2 cells and HUVE cells using a modification of the method of Gubler and Hoffman (10). An EcoRI adaptor was added to the 5’ end of the cDNA by blunt end ligation; the SstI cloning site, encoded by the first strand primer, was exposed by endonuclease digestion. cDNA libraries were constructed by ligating the cDNA to pZEM 228cc, a mammalian expression vector.

The NH,-terminal sequence of the 51-kDa fragment of PLTP allowed the design of degenerate oligonucleotide primers, which led to cloning and identification of a unique 25-base pair nucleotide DNA sequence. An antisense 25-base pair oligonucleotide, as indicated in Fig. 2, was then synthesized and 5’ end labeled with [32PlATP using T4 polynucle- otide kinase. The HepG2 library was titered and suitable dilutions determined to obtain approximately 1.25 x lo6 colonies, which were screened by probe hybridization to bacterial colonies immobilized on nylon filters. The resulting partial cDNA was used to screen a HUVE cell cDNA library.

The PLTP gene was mapped by PCR using DNA from the Human Genetic Mutant Cell Repository Human/Rodent Somatic Cell Hybrid Mapping Panel 2 (National Institute of General Medical Sciences, Co- riel1 Institute for Medical Research). The panel consists of DNA isolated from 24 humadrodent somatic cell hybrids retaining one or two human chromosomes. Specific oligonucleotide primers, sense and antisense 5’ tgattgactccccattgaagctggagc 3’ and 5’ cagctggacctcaggctggtctggtgg 3’ based on the cDNA sequence were generated for this purpose and used for PCR amplification.

A multiple tissue Northern blot (Clontech Laboratories, Inc., Palo Alto, CA) was used and the recommended protocol strictly followed. The multiple tissue blot contained 2 pg each of highly purified poly(A)+ RNA from various human tissues. The Northern blot was probed with a SstI fragment, isolated from the partial PLTP cDNA clone.

Both strands for all templates, including the full-length PLTP cDNA clone, were sequenced using the Applied Biosystems 373A DNA Se- quencer following the procedures provided by the manufacturer for

9388

Human Phospholipid Dansfer Protein cDNA 9389 performing fluorescence-based dideoxy sequencing reactions. All oligo- nucleotide primers for PCR and DNA sequencing were synthesized on an Applied Biosystems model 394 DNA synthesizer. The DNA sequences representing PLTP mRNA were analyzed using software from Intelli- Genetics (Mountain View, CA) and the FASTA package of tools (11). Data base searches for sequences similar to PLTP were conducted in- cluding GenBank (12), EMBL (13). PIR (14), SWISSPROT (15), and the Prosite dictionary of motifs (16). The statistical significance of observed sequence similarity matches was tested by the rdf2 shuffling algorithm (11). 2 values were considered to suggest a significant match when 2 > 10 and probably a significant match when 2 > 6 (17). Oligonucleotide primers were designed using the program Primegen (18) in its single sequence mode.

RESULTS AND DISCUSSION Several investigators have purified and characterized PLTP

showing that it is functionally distinct from CETP (6, 7, 19). In this paper we describe the molecular cloning of human PLTP. Electrophoresis of the active fraction of purified PLTP resolved two bands with molecular masses of 80.6 2 1.1 and 50.6 * 0.6 kDa (Fig. 1). The rabbit antihuman PLTP antibody produced

A B C

97.4 4 66.2

c

45.0 4 I

31.0 Y

21.5 a FIG. 1. Protein gel and Western blot of purified PLTP. Molecular

mass standards are shown in lane A. The final preparation of PLTP was separated by gradient SDS-polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie Blue (lane B) . Western blot of PLTP using the same conditions as in lane B was immunostained with rabbit anti-human PLTP serum (lune C).

against the 51-kDa protein reacted with both the 51-kDa frag- ment and the mature protein as determined by Western blot analysis (Fig. 1). Amino acid sequence analysis of purified PLTP resulted in a 22-amino acid NH2-terminal sequence for the 51-kDa fragment and a 10-amino acid NH2-terminal se- quence for the 81-kDa protein.

The 22-amino acid sequence allowed the design of degenerate primers for PCR which successfully retrieved the correspond- ing portion of the cDNA. Cloning and DNA sequencing of the insert showed that the deduced amino acid sequence was iden- tical to that determined by amino acid sequence analysis. This provided a unique 25-nucleotide DNA sequence. An antisense oligonucleotide probe was prepared from this sequence (Fig. 2) and used to probe a HepG2 library. One positive cDNA clone was found which, when sequenced, was found to be incomplete at the 5’ end. Since Northern blot analysis indicated that PLTP message was present in a wide variety of human tissues, other available libraries were tested. PCR, using specific primers to the known PLTP sequence, showed the presence of PLTP cDNA in HUVE cell and human placenta libraries. A SstI fragment of the partial cDNA obtained from the HepG2 library was then used to screen a HUVE cell library. Eighteen positive clones were confirmed by PCR, and four clones from the HUVE cell library were selected for sequence analysis. The HepG2- and HUVE-derived cDNA clones were identical in their region of overlap (nucleotide 288 to 3’ end).

The complete cDNA sequence, 1,750 base pairs in length, contains an open reading frame of 1,518 nucleotides followed by a 184-nucleotide 3”untranslated region (Fig. 2). Eight out of nine nucleotides were identical to the Kozak sequence (20) flanking the initiation codon. The cDNA does not contain an AATAAA polyadenylation consensus sequence before the site of priming by the poly(T)-containing oligonucleotide, which was present at the 3’ end.

The hydrophobic PLTP signal peptide is composed of 17

GTGGCCGCCGTCGCCCGCATCCCCTCAGCTGCCCGCUTCCUCGTCACCGCGCCGCCCCCUGCTCUCCGCTCAGCCCGCTCGCUTGGCCCTCTTCGGGGCCCTCTTCCTAGCGCTGCTGGUGGCGUUT 135 M A L F G A L F L A L L A C A H 16

G U C A C T T C C U G G C T G C M C l C C G C G T U C C T C ~ G G C G C T G C A G C T G G T W A G U ~ G G G C C T G C G C T T T C T G C A G ~ G A G C T ~ C A C T A T U C U T T C C ~ C C T G C G ~ ~ W A G G C U C T T C 270 A E F P G C K I R V T S K A L E L V K ~ E G L R F L E ~ E L E T I T I P D L R G K E G H F 61

Y Y Y I S E V K V T E L ~ L T S S E L D F ~ P ~ ~ E L M L ~ I T U A S L ~ L R F R R ~ L L 106

Y ~ F F Y D G G Y I Y A S A E G V S I R T G L E L ~ R D P A G R M K ~ ~ N ~ ~ ~ ~ A ~ ~ ~ ~ ~ ~

TACTACMUTCTCTGACCTWAGGTUUCAGCTGCMCTCAUTCTTCCCAGCTCCATTTCUGCUUGU~GCTCATGCTT~TUCCMTGCCTCCTTGCGGCTGC~TTCCGCACAUGCTGCTC 405

TACTGCTTCTTCTATGATGGGGGCTAUTCMCGCCTUGCTCAGGGTGTGTCUTCCGUCTGGTCTGGAGCTCTCCCGGGATCCCGCTGGACGGAT~GTGTCCMTGTCTCCTGCUGGCCTCTGTCTCC 540

AWATCCACGCGGCCTlCGGGG~CCTTCM~GGTGTATCATTTTCTCTCUCGTTUT~CCTUGGGATGCGCTTCCTCCT~CCAGUCATCTGCCCTGTCCTCTACUCGUGGCACGGTCCTGCTC 675 R M H A A F G G T F K K V Y D F L S T F I T S G M R F L L N Q O I C P V L Y H A G T V L L 1%

M C T C C C T C C T G G A U C C G T G C C T G T G C G U G T T C T G ~ G C A C C A G C ~ ~ G ~ ~ G G ~ ~ ~ ~ C ~ A ~ ~ C C C ~ ~ T ~ G C A ~ C C ~ G ~ G G C ~ ~ C ~ C ~ G ~ C C ~ G C A ~ ~ ~ C T T C C G G G G G G C C T T C T T C C C C C T G 810

A C T W C A G W A C T G C A G C C T C C C ~ C C G G C C A G ~ G ~ G C C C ~ ~ ~ G ~ G C A ~ ~ G C G ~ ~ G C ~ G ~ A ~ G ~ G G C C ~ ~ C ~ C ~ C A G ~ ~ C ~ ~ C ~ ~ C C A C ~ C ~ G C ~ ~ ~ C A G C T A C T T C C ~ C G ~ G G C C C T G 945

Y S L L D T V P V R S S V D E L V G I D Y S L M K D P V A S T S N L D M D F R G A F F P L 241

T E R Y U S L P Y R A V E P P L P E E E R M V Y V A F S E F F F D S A M E S Y F R A G A L 286

UGCTGTTGCTGGTGGGGCA~GGTGCCCUCGACCTG~UTGCTGCTCAGGCCUCCTACTTTGGCAGUTTGTCCTGCTCAGCCUGUGTCATTCACTCCCUTT~GCT~GCTGC~TCCTGCCC 1080 ~ L L L ~ ~ D K ~ P H D L D ~ L ~ R A ~ Y F G ~ I ~ L L S P A ~ I D ~ P L K L E L R ~ L A 331

C U C C G C G C T G U C U T C M ~ C C T C T G G U C U C U T C T C T G T ~ C ~ G C ~ A G C G ~ ~ C ~ ~ ~ G C C C ~ ~ T C C ~ C ~ C A C C A G C C ~ C A G G ~ C ~ G C ~ G ~ C ~ G ~ ~ C A C ~ A ~ ~ C G C C C G T C T U G C G C C M G 1215 P P R C T I K P S G ~ T I S V T A S V T I A L V P P D ~ P E V ~ L S S M T M D A R L S A K 376

ATCGCTCTCCGCGGWAGGCCCTGCGUCCCAGCTGGACCTGCGUGGTTCCWATCTATTCCMCUTTCTGUCTGCAGTCGCT~TCTCATCCUTTAUGCCCCCTCTWACACUTGCT~CATT~ 1350 M A L R G K A L R T ~ L D L R R F R I Y S Y H S A L E S L A L I P L ~ A P L K T M L ~ I G 421

GTCATGCCUTGCTCMTCAGCGCACCTGGCGTGGGGTGUCATCCUCTACCTCAGGGUTCMCTTTGTGUTCAGGTGGTCACWACUTGCGGCATTCCTUCUTCGCGCCTCATCTCUCTTTGC~ 1485 V M P M L Y E R T U R G V ~ I P L P E G I Y F V H E V V T N H A G F L T I G A D L H F A K 466

G G G C T C C C A W G C ~ C A T T C A ~ ~ C C G G C C ~ G C ~ C A ~ G ~ ~ G G G C G ~ C ~ C ~ G C C C C ~ ~ C C G ~ C ~ ~ G ~ G C T G ~ C ~ C A G C C C ~ ~ ~ C C C ~ G C ~ G G ~ G C ~ G ~ ~ ~ ~ ~ G C A C C C ~ C C C C T C T U 1620

GCCCCTCTTTTCCUUTTUTA~CTGTAGT~CCCC~CTMCCCCUGTGCUUCA~GACGGCATTT~GCTGTACC~TTTMTTCUTMTCMTCTATCMTTAUGTCCGTCUCUCC 1750

FIG. 2. Nucleotide sequence of PLTP cDNA and derived amino acid sequence of PLTP. Nucleotide numbering starts at the 5‘ end and is indicated by the numbers a t the end of each line. The open reading frame extends from nucleotide 49 to 1566. The stop codon is indicated as a period. Translation is shown using the one-letter code directly underneath the second base of each codon. The signal peptide is numbered 1-17. The mature protein is numbered consecutively starting at amino acid 18 and ending at amino acid 493. The Kozak sequence is found a t nucleic acid positions 83-91. The location of the NH2 terminus of the 51-kDa proteolytic fragment of PLTP, determined by amino acid sequence analysis, extends from amino acid 163 to 184. Putative N-linked glycosylation sites are located at amino acids 64, 94, 117, 143, 245, and 398. The five extended hydrophobic domains predicted by the algorithm of Eisenberget al. (38) are located a t amino acids 1-21,105-125,184-203.299-319, and 405-425. There are seven potential protein kinase C phosphorylation sites, located a t residues 27, 124,160,242,336,374, and 430. Four cysteines are found in the mature protein a t amino acids 22, 146, 185, and 335. The antisense oligonucleotide probe used to screen the HepG2 library corresponds to nucleic acids 597-621. The SstI fragment used to screen the HUVE library corresponds to nucleic acid 481 and extends to the 3’ end of the cDNA. The cDNA terminated in 18 A nucleotides, which were not included in the figure.

G L R E V I E K Y R P A D V R A S T A P T P S T A A V . 493

9390 Human Phospholipid 'IFansfer Protein cDNA

1. 2 . 3 . 4 . 5 .

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3 . 4 . 5 .

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1. 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 .

- - - - - - - - HALFGALFLALLAGAHA--------EFPGCKIRVTSKALELVKQESLQSGLRFLEQELETITIPDLRQ-----KEQHFYYNISEVKVTELQLTSBELDFQPQQELUL - - - - - - - - HLMTVLTLALLGNAHACSKGTSHEAQIVC--RITKPALLVLNHETAKVIQTAFQRASYPDITQEKAHULLQQVKYGLHNIQISHLSIASBQVELVEAKSIDV """""""""""" - - - - - - - - ACPKGASYEAQIVC--RITKPALLVLNQETAKWQTAFQRAGYPDVSQERAVMLLQRVKYGLHNLQISHLSIASSQVELVDAKTIDV

HLMTVLTLALLGNVHACSKGTSHKAQIVC--RITKPALLVLNQETAKVIQSAFQRANYPNITQEK~MLLQQVKYGLHNIQISHLSIASSRVELVEAKSIDV UGALARALPSILLALLLTSTPEALGANPQLVA--RITDKGLQYMQEGLLALQSELLRITLPDFTQDLRIPHVQRGRYEFHSLNIHSCELLHSALRPVPGQGLSL

HGTWARALLGSTLLSLLLAPGALGTNPQLIT--RITDKGLEYMREGLLALQRKLLEVTLPDSDQDFRIKHFQRAQYKFYSLKIPRFELLRGTLRPLPGQGLSL HRENHARGPCNAPRWVSLHVLVAIGTAVTMVNPQ~--RISQKGLDYASQQGTMLQKELKRIKIPDYSDSFKIKHLQKGHYSFYSUDIREFQLPSBQISMVPNVGLKF

""" ""_ "" HARGPDTARRWATLWLMLGTAVTT-TNPQIVA--RITQKGLDYACQQGVLTLQKELEKITIPNFSQNFKIKYLQKGQYSFFSMVIQGFNLPNSQIRPLPDKGLDL

QITNABL---QLRFRRQLLYWFFYDGGYINASAE-GVSIRTGLELSRDPAGRUKVSN-VSCQASVSRMHMFGQ--TFKKVYDFLSTFITSGMRFLLNQQICPVLYHAGTV SIQNV8WFKQTLKYGYTTA~LGIDQSIDFEIDSAIDLQINTQLTCD-SGRVRTDA-PDCYLSFHKLLLHLQQEREP~IKQLITNFISFTLKLVLKGQICKEI-NVIBN AIQNV8WFKQTLNYSYTSAWGLGINQSMFEIDSAIDLQINTELTCD-AGSVRTNA-PDCYLAFHKLLLHLQQEREPOVLKQLITNFISFTLKLILKRQVCNEI-NTISN SIQNVSWFKQTLKYGYTTAWGLGIDQSMFEIDSAIDLQINTQLTCD-SGRVRTDA-PDCYLSFHKLLLHLQQEREPOVIKQL?TNFISFTLKLVLKGQICKEI-NIISN SISDSSIRVQQR~---VRKSFFKLQGSFDVSVK-GISISVNLLLGSE-SSGRPTGYCLSCSSDIADVEVDMSQ--DS~LLNL?HNQIESKFQKVLESRICEMIQKSVSS DISDAYIHVRQSWIC---VRKAFLRLKNSFDLYVK-GLTISVHLVLGSE-SSGRPTVTTSSCSSDIQNVELDIEG--DLEELLNLLQSQIDARLREVLESKICRQIEEAVTA SISNANIKISQKWIC---AQKRFLKUSGNFDLSIE-GMSISADLKLGSNPTSGKPTITCSSCSSHINSVHVHISK-SKVQHLIQLIHKKIESALRNKMNSQVCEKVTNSVBS SIRDASIKIRQKWIC---ARKNFIKLGGNFDLSVE-GISILAGLNLGYDPASGHSTVTCSSCSSGINTVRIHISQ-SSL~LIQLIRKRIESLLQKSMTRKICEWTSTVSS

6 4 1 EFELNDEKMTRM-APNOVKSRL?RHSTQKNLK---NSQMCQKSLDVETD

DLQPYLQTLPVTT--EIDSFADIDYBLVEAPRATAQMLEVHFKQEIFHRNHRSPVTLLA----MEEHNK~--YFAIBDYV-INTASLVYHEEQ-YLNFSITDDHIPPDS IHADFVQTRMSI--LSDGDIQMISLTGDPIITASYLESHHKQYIIYKNVSEDLPLPTFS-PALLGDS~L--YFWFSEQV-~HSLAKVAFQDQ-RLMLSLUGD---EFK

HLQPYLQTLPVTT--QIDSFAQIDYSLUEAPRATAGMLDVMFKQEIFPLDHRSPVDFLAPAHNLPEAHSRWV"YFSIBDYV-INTASLAYHKSQ-YWNFSITDAHVPADL KLQPYFQTLPVMT--KIDSVAQINYGLVAPPATTAETLDVQMKQE?YSENHHNPPPFAPPVMEFPMHDRMV--YLGLBDYF-?NTAGLVYQEAQ-VLKUTLRDDMIPKES KLQPYFQTLPVTT--KLDKVAQMYSLVAPPRAT~NLDWLLKGE?FSLAHRSPPPFAPPALAFPSDHD~--YLGISEYF-INTAGFVYQKAQ-ALNLTLRDDMIPKES GLEANVPPVSFLKVLKLNKTEWPYFWGTVCAIANGGLQPAF-SVIFSEIIAIFGPGDDAVKQQKCNIFSLIFLFLGIISFFTIFLQGFTFGKAQEILTRRLRS-MAFK~

DHLLRATY?GSIVLLSPAVIDSP-LK-LELRVLAPPRCTIKPSGTTISVTASVTIALV--PPDQPEVQLSSUTMDARLSAKMALRGKALRTQLDLRRFRIYS--------- AVLETWGFNTNQEIFQEWGGFP-SQ-AQVTVHCLKMPKISCQNKG~SSVMVKFLFPRPDQQHSVAYTFEEDIVTTVQASYSKKKLFLSLLDFQITPK-----""- KVLETQGFDTNQEIFQELSRGLPTGQ-AQVAVHCLKVPKISCQNRG~SSSVAVTFRFPRPDGREAVAYRFEEDIITTVQASYSQKKLFLHLLDFQCVPASGRAGSSANL AVLETWGFNTNQEIFQEWGGFP-SQ-AQVTVHCLKUPRISCQNKGYYVNSSVHVKFLFPRPDQQHSVAYTFEEDIMTTVQASYSKKKLFLSLLDFQITPK---------- NIRLTTKSPRPFVPRLARLYPNMNLE-LQGSVPSAPLLNFSPGNLSVDPYMEIDAFVLLPSSSKEPVFRLSVATNVSATLTFNTSKITGFLKPGKVKVELK---------- NIRRTTKS?RPFVPLLANLYPNMNLE-LQGT~SEQLVNLSTENLLEEPEMDIEALWLPSSAREPVFRLGVATNVSATLTLNTRKITGFLKPGRLQVELK---------- KFRLZTKF?GTFLPEVAKKFPNMKIQ-IHVSASTPPHLSVQPTGLTFYPAVDVQAFAVLPNSSLASLFLIGUHTTGSUEVSAESNRLVGELKLDRLLLELK---------- KFRLTTKF?GILIPQVAKMFPDMQHQ-LFIWASLPPKLTMKPSSLDLIFVLDTQAFAILPNSSLDPLFLLEUNLNLS~GAKSDRLIGELRLDKLLLELK---------- -LRQDMSWFDDHKNSTGALSTRLATDMQVQGATGTRLALIAQNIANLGTGIIISFI---YGWQLTLLLLAWPIIAVSGIVEMKLLAGNAKRDKKELEA-----------

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1 0 1 9 0

8 5

1 0 3 1 0 1

103 109 104

2 0 9 194

2 0 9 1 9 3

2 0 7 2 0 7 2 1 5 2 1 0 6 8 6

2 9 9 3 1 0 294 3 1 0 3 0 8 3 1 2 3 2 0 3 1 5 7 9 5

3 9 7 4 0 9 4 04 4 0 9 4 0 8 4 1 2 4 2 0 4 1 5 8 9 1

4 9 3 4 9 3 4 9 7 4 9 3 4 7 1 4 8 1 4 8 7 4 8 2 9 5 7

sequences of 1, PLTP-human; 2, CETP-human; 3, CETP-rabbit; 4, CETP-cynomolgus monkey; 5, LBP-human; 6, LBP-rabbit; 7, BPI-human; 8, FIG. 3. Multiple alignments of PLTP with amino acid sequences of several proteins. Shown is one possible multiple alignment of the

BPI-bovine; and 9, amino acids 641-957 of the P-glycoprotein encoded by the human multidrug resistance protein 3 gene. The bold letters in the figure indicate areas where at least 6 amino acids are identical.

amino acids, ending in Ala-His-Ala, a frequently occurring tri- plet preceding signal peptide cleavage (21). Interestingly, both the PLTP and CETP leaders are 17 amino acids in length with 9 identical amino acids. Similarity among the signal sequences of CETP, lipoprotein lipase, apolipoprotein A-I, and apolipopro- tein A-IV has been described (22).

The mature protein is comprised of 476 amino acids with a predicted molecular mass of 54,719 kDa. Mature CETP has an identical number of amino acids. The deduced NH2-terminal sequence of PLTP was identical to the NH2-terminal sequence of the 81-kDa protein determined by amino acid sequence anal- ysis. There are six potential N-glycosylation sites predicted from the PLTP sequence, which may account for the discrep- ancy between the calculated molecular mass (55 kDa) and the mass estimated by SDS-gel electrophoresis (81 kDa). CETP and PLTP both are hydrophobic proteins with numerous hydropho- bic regions found throughout their protein structures.

Sequences that scored highly in similarity searches of avail- able sequence data bases with PLTP included human (3) cyno- molgus monkey (231, and rabbit (24) CETP, all about 20% iden- tical to PLTP. PLTP also scored highly in comparisons with human and rabbit lipopolysaccharide-binding protein (LBP) (25) (with an identity of 24 and 26%, respectively) and with human (26) and bovine (27) bactericidal permeability-increas- ing protein (BPI) (with an identity of 26 and 27%, respectively) (Fig. 3). Further sensitive data base searches with various members of this family revealed that human multidrug resist- ance protein 3 exhibited a probable significant similarity (28).

Individual local sequence comparisons were conducted be- tween PLTP and a number of sequences that might contain similar functional motifs. PLTP residues 163-203 are 23% identical to residues 264-309 of human phospholipase C (29).

9.50 - 7.50 - 4.40 -

FIG. 4. Northern blot analysis of poly(A)+ RNA from various human tissues. Two pgllane of poly(A)+ RNA from various human tissues (Multiple Tissue Northern Blot, Clontech) was probed with a 1,287-base pair SstI fragment isolated from the HepG2 cDNA clone that was random primer labeled. The order of samples on the blot is as follows: lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; and lane 8, pancreas.

pholipase C, which starts at residue 312. PLTP is distinct from a heterogeneous class of smaller intracellular phospholipid transfer proteins (30).

Hybridization of a PLTP probe to poly(A)+ mRNA isolated from various human tissues indicated the existence of a single transcript of approximately 1.8 kilobases for each tissue, con- sistent with the length of 1.77 kilobases for the cDNA (Fig. 4). The following tissues had detectable levels of PLTP mRNA in decreasing order: placenta 2 pancreas > lung > kidney > heart > liver > skeletal muscle > brain. Also, total mRNA prepared from microvascular, arterial, and venous derived human endo- thelial cells showed a single transcript of approximately 1.8

This region is just upstream of the catalytic domain X of phos- kilobases (data not shown).

Human Phospholipid Dansfer Protein cDNA 9391

DNA isolated from 24 humadrodent somatic cell hybrids were used to map the PLTP gene to chromosome 20. LBP and BPI share significant functional and structural similarity and have also been mapped to chromosome 20, between q11.23 and q12 (31), whereas CETP is located on the long arm of chromo- some 16 (32).

CETP and PLTP have related functions, and previous studies suggest that they play coordinated roles in lipid transfer (33, 34). Evidence indicates that CETP is involved in the process of HDL interconversion (35), and increased CETP activity has been shown to have a proatherogenic effect (4, 36, 37). The similarities between PLTP and CETP suggest that PLTP might also play a role in atherogenesis. The identification and cloning of human phospholipid transfer protein will greatly facilitate structural and functional studies and the elucidation of the role of PLTP in lipoprotein metabolism and pathophysiology.

Acknowledgments-We thank Dr. Theodore Whitmore for the chro- mosome localization work, Erica Vanaja for homology searches, Simon Evans for amino acid sequence analysis, William Lint for oligonucle- otide synthesis, and Dr. William Pearson for computer software.

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