Cloning, sequence comparison and in vivo expression of the gene encoding rat P-selectin

Gene, 145 (1994) 251-255 0 1994 Elsevier Science B.V. All rights reserved.

GENE

251

0378-l 119/94/$07.00

08004

Cloning, sequence comparison and in vivo expression of the gene encoding rat P-selectin (Adhesion molecules; inflammation; lectin; endotoxin)

John A. Auchampach”, Mary G. Oliverb, Donald C. Andersona and Anthony M. Manning” “Adhesion Biology, Upjohn Laboratories, Kalamazoo, MI 49001 USA; and bHospitalfor Sick Children, Toronto, Ontario, M5G 1X8, Canada. Tel. (l-416) 813-1500 Received by CM. Kane: 22 October

1993; Revised/Accepted:

28 February

1994; Received at publishers:

7 April 1994

SUMMARY

We have cloned the cDNA encoding rat P-selectin (Psel) and have examined the regulation of Psel expression in vivo. Sequence analysis of the complete Psel cDNA demonstrated significant nucleotide and amino-acid identity with human and mouse Psel. Similar to mouse Psel, the rat sequence lacks the equivalent of human complement regulatory protein-like repeat 2 (CR2). Seven potential N-linked glycosylation sites are conserved between the three species, suggesting that carbohydrate modification may play an important role in Psel function. To examine expression of Psel in vivo, levels of Psel mRNA were examined in several different tissues after systemic administration of lipopolysaccharide (LPS). Psel mRNA was undetectable in tissues of vehicle-treated animals. By 3 h after LPS administration, Psel mRNA levels were elevated in all tissues examined, the highest levels being seen in the lung. Significant increases in Psel mRNA were also seen in the heart, thymus, spleen and kidney. By 24 h after LPS, mRNA levels for Psel remained elevated in the lung, heart, kidney, thymus and small intestine. Psel mRNA was not detectable in total RNA isolated from purified rat platelets, suggesting that the increased levels of Psel mRNA were the result of upregulation of endothelial gene expression. In addition, only minimal levels of platelet factor 4 mRNA (PF4), used as a platelet-specific marker, were observed in the tissues studied. These data demonstrate that part of the response to acute inflamation in vivo includes the rapid increase in endothelial Psel expression.

INTRODUCTION

P-selectin (Psel) is a member of the selectin family of cell-surface adhesion molecules (McEver 1991). This integral membrane glycoprotein is found in Weibel-Palade Correspondence to: Dr. A.M. Manning, Upjohn Laboratories, 385-5450; Fax (l-616)

Kalamazoo, 384-9308.

Adhesion MI

49001,

Biology, USA.

7238-267-3, Tel.

(l-616)

Abbreviations: aa, amino acid(s); bp, base pair(s); CR (see Fig. l), complement protein-like regulatory repeat(s); EGF, epidermal growth factor; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; kb, kilobase(s) or 1000 bp; LPS, lipopolysaccharide; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PF, platelet factor; Psel, P-selectin; Psel, gene (DNA) encoding Psel; SDS, sodium dodecyl sulfate. SSDI 0378-1119(94)00215-E

bodies of endothelial cells (Bonfanti et al., 1989; McEver et al., 1989) and in c1granules of platelets (Hsu-Lin et al., 1984; McEver et al., 1984). Upon stimulation of these cells with various agonists Psel is rapidly mobilized to the cell surface where it supports the binding of neutrophils and monocytes, mediating their interaction with endothelium or platelets located on the vessel wall (Larsen et al., 1989; Geng et al., 1990). In addition to this immediate response, pro-inflammatory cytokines can also elicit Psel gene expression and prolonged expression of Psel on the endothelial cell surface (Weller et al., 1992). Psel has been suggested to mediate capture of leukocytes from the circulation to the vessel wall at high shear rates, leading to the phenomenon of leukocyte rolling (Lawrence and Springer, 1991). This process is thought to be the

252 first step in the emigration tion into i&lammed strated

to mediate

monocytes

within

be important

of leukocytes

tissues.

the accumulation thrombi,

from the circula-

Psel has also been demonof neutrophils

a process

for fibrin deposition

which appears

(Palabrica

The aim of this study was the cloning in a rat model

to

ct al., 1992).

and sequence

sis of the rat cDNA for Psel, and the examination gene expression

and

cDNA

sequence

domain

1991), consisting to a family EGF-like

analy-

repeated

Psel between

of an N-terminal structure,

and aa sequence analysis of rat Psel:

comparison with human and mouse Psel The nt sequence

analysis

that the entire sequence

is 3 185 bp in length with an ORF

homologous

followed

a series

by an

of tandemly-

aa identity

to human

of rat and mouse

Psel

et al., 1991) is shown. The most highly conserved

regions

included

the

lectin,

suggesting

further supports

EGF-like.

and

the

cyto-

that these regions are impor-

The high degree of conservation domains

structure-function

the critical role of these domains

of the rat Psel cDNA revealed

similar ( McEvcr.

(Sanders

of the lectin and EGF-like (a) Nucleotide

lectins,

and

each of the domains

tant for Psel function.

AND DISCUSSION

displaying

domain

CR (Fig. 2). The percent

plasmic domains, EXPERIMENTAL

a protein

to that of other selectins

of Ca’+ -dependent repeat

of Psel

of acute inflammation.

predicted

organization

between

these species

studies demonstrating in ligand binding

(Geng

et al., 1992). Disdier

et al. (1992) have demonstrated

that the cyto-

of 2304 bp. The 3’ untranslated region of 862 bp is greater than that reported for human (614 bp) and mouse

plasmic domain encodes those signals for sorting of Psel into secretory granules. The conserved aa sequence of the

(128 bp) Psel. The complete cDNA sequence of rat Psel encoded a polypeptide of 768 aa that displayed 75% overall sequence identity with human Psel (Fig. 1). The

cytoplasmic tail between species supports this hypothesis (Fig. 1). Compared to the human sequence, there are four identical aa substitutions in this region in the rat and

I--+

Lectin t MANCQIAIL;QRFQRWFG;SQLLCFSAL;SELTNQKEVAAWTYHYSTK;\YSWNISRKYCQNRYTDLVA;QNKNEIDYLNKVLPYYSSY;WIGIRKNNK;WTWVGTKKALTNEAENWADN __G_PKGSWTP_LRS_~L-GA--IW------__V__________N_________N__VF_RRRF~~_~________AH__D_~_F~N________I__________N_T-_~--__----

120 120

,,CRZ r)CR3. KLECLASGIWTNKPPQCLA;\QCPPLKIPERGNMICLHSAKAFQHQSSCSFSCEEGFALV;;PEWQCTAS;;VWTVQCQHLEAPSEGTMDCVHPLTAFAYGSSCKFECQPGYRV _D-----S----PH---A-M--IA-_--D_-__-______-A E-Q---------N--K.............................................................. _K_____S_-__LH___D_T---A____D____________M EMQ---------N--Q..............................................................

360 298 298

t

.*

WGNFSYGSICSFHCLEGQLLNGSAQTACQENGHWSTTVPTCQAGPLTIQEALTYFGGAVASTIGLIMGGTLLALLRKRFRQKDDGKCPLNPHSHLGTYG~TN~FDPTP

Fig. 1. Comparison

of human

(H), mouse (M) and rat (R) Pscl aa sequences.

Domain

boundaries

are identilied

with arrows

830 Human

with the numbermg

of

CR as in the human. Residues encoding the transmembrane domain are denoted with a thick overline (-). Potential N-linked glycosylation sites conserved between human, mouse and rat Psel are indicated by asterisks (*). Methods: A cDNA library was prepared from a combination of LPStreated rat lung and spleen mRNA in the hZAPI1 phage vector (Stratagene, La Jolla. CA, USA). The unamplified cDNA library was scrcencd by plaque filter hybridization with a canine Psel cDNA (Manning et al., 1992) as a probe using standard procedures (Sambrook et al.. 19X9). A single cDNA clone was identified and purified before plasmid rescue in pBluescriptI1 SK(-) using the in vivo excision method (Short et al.. 198X). The complete DNA sequence of both strands of this clone (RG 1D) was determined using the dideoxynucleotide chain-termination method (Sanger et al.. 1977). This sequence contained 2060 bp which demonstrated significant sequence identity to human and mouse P-selectins. The remaining 5’ end of rat Psel was obtained using reverse transcription and PCR. A gene-specific primer antisense to a region at the 5’ end of RGlD was designed and reverse transcriptase (Gibco BRL. Grand Island, NY. USA) was used to synthesize cDNA from total cellular RNA isolated from LPS-stimulated rat heart tissue. Subsequently, the cDNA was amplified by PCR (30 s at 94 C. 30 s at 55 C and 1 min at 72 C for 35 cycles) using a nested antisense gene-specific primer (5-ATTTGCAACGCTTCACAGACTG) and a 5’ sense homology primer (5’-CCAGMMGARRTCASAGAGGASATGGC) which was designed based on the conserved sequence of murine and human Psrl within the region of the signal sequence (M, A or C: R. A or G; S. C or G). Following agarose gel purification, the resulting fragment was subcloned into pBluescriptI1 SK(+) and three independent clones from two PCR reactions were sequenced. This sequence has been submitted to GenBank and has been assigned the accession No. L23088.

253 "LECTIN"DOMAlN SIGNALTDE

\

"EGFjmMAIN

TRANSMEMBRANE "C3B-C4BREGULATORYPROTEIN"REPEATS

I

CYTOPLASMIC TAIL

\

1 H

M NH2 % identity

Fig.2. The domain species. The absence between

56

76

%identity

45

structure

of human

of the equivalent

90

77

89

70

12

3

4

5

6

7

8

9

79

61

61

80

19

61

12

(H), mouse (M) and rat (R) Psel. The % aa identities of human

CR2 in mouse

to human

and rat Psel have been determined

83

89

H 78

is displayed to maximally

83 below each domain align

homologous

for each sequences

species.

sequences: Phe737-+ Leu, Gl~‘~~+Lys, mouse Phe765-+Tyr and Ser 768+Thr. These substitutions would be predicted to represent conservative aa substitutions. Psel itself is internalized after its appearance on the cell surface and a candidate endocytosis signal, YGVF, is conserved in all of the species. The number of CR in rat Psel was found to differ from that seen with human (Figs. 1 and 2). Human Psel is known to contain nine CR, whereas the rat and mouse sequences contain only eight, lacking the equivalent of CR2. It has been suggested that the function of the CR is simply to serve as structural scaffolds to facilitate the efficient interaction of the lectin and EGF-like domains with their counter-receptor (McEver, 1991). The observation that various CR are excluded in certain species supports this concept. There are nine potential N-linked glycosylation sites present in the rat Psel polypeptide having the concensus sequence NXS/T (Fig. 1). All are located on the extracellular portion of the molecule. As compared to the human and murine sequences, seven of these potential glycosylation sites are present at identical positions. The conservation of these seven N-linked glycosylation sites between human, murine and rat Psel suggests that carbohydrates structures on the Psel molecule may be important for Psel function. (b) Regulation of Psel mRNA expression in vivo To evaluate the regulation of Psel expression in vivo, we examined levels of Psel mRNA in tissues of animals administered LPS. Psel mRNA was not detected in tis-

sues of vehicle-treated control animals. However, 3 h after LPS administration, Psel mRNA levels were elevated in several different tissues, most notably in lung, heart,

kidney, thymus and spleen, but including brain, liver and small intestine (Fig. 3a). Low levels of Psel mRNA were detected in heart, kidney and small intestine 24 h following endotoxin administration. In the lung, the Psel message remained significantly elevated 24 h following endotoxin. The differences in Psel mRNA levels observed between the different tissues may be due to diffeences in the degree of vascularization of these tissues or perhaps to differences in tissue-specific responses of endothelium in these tissues to LPS. Since it has been reported that small amounts of Psel mRNA can be detected in poly(A)+RNA isolated from platelets (Johnston et al., 1989) and because LPS may promote aggregation and accumulation of platelets within organs, we wished to determine whether the increase in Psel mRNA observed in LPS-treated tissues was the result of platelet accumulation. To accomplish this, we examined levels of the platelet-specific mRNA PF4 (Poncz et al., 1987) in tissues of animals administered LPS. PF4 mRNA was detectable only in spleen and lung tissue of vehicle-treated animals (Fig. 3b). Slightly increased levels of PF4 mRNA were observed in spleen and lung tissues 3 h following LPS. Little PF4 message was observed at 24 h. To ensure that PF4 mRNA is contained within platelets, total cellular RNA was isolated from purified rat platelets and levels of PF4 and Psel mRNA were analyzed by Northern blotting. Psel mRNA was undetectable in total RNA from platelets, although a significant amount of PF4 mRNA is present. Taken together, these results suggest that the increase in Psel mRNA observed in LPS-treated rat tissues is not the result of platelet deposition but is more likely the result of induced expression in endothelial cells. Weller et al. (1992) have demonstrated that Psel tran-

P-Se1 -

28s

-

18s

Control

LPS (3h)

LPS (24W P-Se1

PF4

Fig. 4. Psrl and PF4 mRNA

Etd Br

levels in LPS-treated

lung tissue and tn

platelets. Methods: Total RNA isolated from lung tissue obtained from rats treated with LPS for 3 h and from rat platelets purilicd from normal animals were subjected to Fig. 3.

to Northern

analysis

as described

in the legend

PF4 Control

LPS (24h)

scription can be induced in murine endothelial cell lines in vitro in response to LPS and that this results in expression of Psel on the cell surface. We hypothesize that the elevated levels of Psel mRNA observed in the rat tissues studied here will result in the elevated expression of Psel on the endothelial cell surface. This Psel may play a role in the accumulation of neutrophils that occurs in tissues following LPS administration. Of note, this accumulation of neutrophils was maximal in the lung (as determined by increased myeloperoxidase activity; data not shown) where Psel mRNA levels were highly elevated. Further experiments utilizing anti-Psel monoclonal antibodies in this model will determine if elevated expression of Psel plays a role in the accumulation lung.

of neutrophils

in the

G3PDH

100 mg of tissue was removed

and immediately

frozen in liquid nitrogen.

Total RNA was isolated by the acid-guanidinium-phenol chloroform procedure (Chomczynski and Sacchi. 1987). RNA (10 pg) was electrophoresed on 1% agarose gels containing 1% formaldehyde and then transfered to a nylon membrane (Genescreen Plus, DuPont) by capillary transfer. The membranes were hybridized in Quikhyb rapid hybridization buffer (Stratagene) containing approx. 1 x 10” cpmiml randomhexamer-labelled cDNA probe at 65°C for 2 h. The rat Pxl cDNA

LPS (24h) Fig. 3. Psel (A), PF4 (B) and G3PDH (C) mRNAs in various tissues following systemic administration of LPS. Methods: Male SpragueDawley rats were administered Salmanellu abortus qui endotoxin (5 mg/kg intraperitoneal) 3 or 24 h prior to killing. Control animals were administered an equal volume of vehicle. At killing, approx.

probe consisted of a 880-bp fragment obtained by PCR from the tsolated cDNA clone and the PF4 probe consisted of a 300-bp fragment obtained by reverse transcription and PCR of total RNA isolated from purified rat platelets. Rat platelets were isolated according to the procedure of Dore et al. ( 1993). Filters were washed with 2 x SSC at 60’C for 15 min and with 2 x SSC/O.S% SDS for 15 min at 6OC and then were exposed to Amersham Hyperfilm MP. SSC is 0.15 M NaCl!0.015 M Nacitrate pH 7.6.

255

(c) Conclusions (1) We have cloned and sequenced the cDNA for rat Psel. The entire nt sequence consists of 3185 bp with a coding sequence of 2305 bp. (2) The aa sequence analysis of rat Psel demonstrates that this 768 aa polypeptide is highly homologous to human and mouse Psel, and that the rat and mouse Psel sequences are missing the equivalent of human CR2. (3) Seven potential N-linked glycosylation sites are conserved between the three known sequences, suggesting that glycosylation at these sites may be important for function. (4) Administration of LPS to rats resulted in marked elevation of endothelial Psel mRNA levels in several tissues, most notably lung. This suggests that Psel transcription may play an important role in the inflammatory response.

from selectins interact

with both cell surface ligands

Hsu-Lin,

S.-C, Berman,

CL., Furie, B.C., August,

bin-activated Johnston,

platelets.

J. Biol. Chem. 259 (1984) 9121-9126.

G.I., Cook, R.G. and McEver,

granule

membrane

similarity

protein

to proteins

R.P.: Cloning

of platelets

involved

in cell adhesion

a

Sequence

and inflammation.

Cell 56 (1989) 103331044. Larsen,

E., Celi, A., Gilbert,

R., Wagner, mediates

G.E., Furie, B.C., Erban,

D.D. and Furie, B.: PADGEM

the interaction

of activated

protein:

platelets

monocytes. Cell 59 (1989) 3055312. Lawrence, M.B. and Springer, T.A.: Leukocytes physiologic through

flow rates: distinction

integrins.

Manning,

W.E., Michael,

J.K.., Bonfanti, A receptor

that

with neutrophils

and

roll on a selectin

from and prerequisite

at

for adhesion

Cell 65 (1991) 8599873.

A.M., Kukielka,

G.L., Dare,

L.H., Entman,

M., Hawkins,

M.L., Smith,

H.K.,

Sanders,

C.W. and Anderson,

D.C.: Regulation of GMP-140 expression in a canine model of myocardial inflammation. FASEB J. 7 ( 1992) A1060. R.P. and Martin,

McEver,

R.P., Beckstead,

Bainton,

M.N.: A monoclonal

J.H., Moore,

D.F.: GMP-140,

antibody platelets.

to a mem-

J. Biol. Chem.

K.L., Marshall-Carlson.

a platelet

alpha-granule

L. and

membrane

pro-

tein, is also synthesized by vacular endothelial cells and is located in Weibel-Palade bodies. J. Clin. Invest. 84 (1989) 92-99. McEver,

R.P.: Novel receptors

that mediate

leukocyte

adhesion

inflammation. Thromb. Haemost. 65 (1991) 2233228. Palabrica, T., Lobb, R., Furie, B.C., Aronovitz, M., Benjamin, Y.-M., Sajer, S.A. and Furie, B.: Leukocyte

accumulation

fibrin deposition is mediated in vivo by P-selectin lets. Nature 359 (1992) 848-850. Poncz,

M., Surrey,

Conway, platelet

REFERENCES

of GMP-140,

and endothelium:

brane glycoprotein binds only to activated 259 (1984) 979999804.

The authors wish to thank J.L. Slightom and R.F. Drong for assistance with DNA sequencing, and H. Jaeschke and CA. Simmons for assistance with animal experiments.

D. and Furie, B.: A

platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for throm-

McEver,

ACKNOWLEDGEMENTS

and Cal+ ions.

J. Biol. Chem. 267 (1992) 19846-19852.

S., LaRocco,

T.M. and Schwartz, factor 4 cDNA

P., Weiss, E.: Cloning

during C., Hsu,

promoting

on adherent

plate-

M.J., Rappaport,

E.F.,

and characterization

derived from a human

of

erythroleukemic

cell

line. Blood 69 (1987) 219-223. Bonfanti,

R., Furie,

(GMP-140)

B.C., Furie,

is a component

B. and

Wagner,

of Weibel-Palade

D.D.:

bodies of human endo-

thelial cells. Blood 73 (1989) 1109-1112. Chomczynski, P. and Sacchi, N.: Single step method by acid

guanidinium

domain

of P-selectin

for sorting into the regulated (1992) 3099321. Geng, J.-G., Bevilacqua,

of RNA isolation

thiocyanate-phenol-chloroform

Anal. Biochem. 162 (1987) 156-159. Disdier, M., Morrissey, J.H., Fugate, R.D., Bainton, R.P.: Cytoplasmic

PADGEM

secretory

M.P., Moore,

extraction.

Nature

Geng, J.-G, Heavner,

(CD62) contains pathway.

the signal

Mol. Biol. Cell 3

K.L., McIntyre,

T.M., Prescott,

R.P.: Lectin domain

peptides

E.F. and

Maniatis,

T.: Molecular

Cloning.

Laboratory

A

Press,

Sanders, E.W., Wilson, R.M., Bahantyne, C.M. and Beaudet, A.L.: Molecular cloning and analysis of in vivo expression of murine Blood 80 (1992) 7955799.

Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Nat]. Acad. Sci. USA 74 (1977) 5463-5468. Short,

343 (1990) 7577760. G.A. and McEver,

J., Fritsch,

Laboratory Manual, 2nd ed. Cold Spring Harbor Cold Spring Harbor, NY, 1989.

P-selectin. D.F. and McEver,

S.M., Kim, J.M., Bliss, G.A., Zimmerman, G.A. and McEver, R.P.: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140.

Sambrook,

J.M., Fernandez,

J.M., Sorge,

J.A. and

Huse,

W.D.:

Lambda

ZAP: A bacteriophage expression vector with in uiuo excision properties. Nucleic Acids Res. 16 758337592. Weller, A., Isenmann, S. and Vestweber, D.: Cloning of mouse endotheha1 selectins. Expression of both E- and P-selectin is inducible by tumor

necrosis

factor. J. Biol. Chem. 267 (1992) 15176-15183.