321
Biochimica et Biophysica Acta, 1131 (1992) 321-324 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90374
Short Sequence-Paper
Sequence and expression of a rat c D N A for LECAM-1 Toshiki Watanabe a , b Yuru Song ", Youko Hirayama c, Takuya Tamatani Keisuke Kuida b and Masayuki Miyasaka b
b
a Department of Pathology, Institute of Medical Science, The University of Tokyo, b Department of Immunology, Tokyo Metropolitan Institute for Medical Science and c Department of Viral Oncology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo (Japan) (Received 14 April 1992)
Key words: Adhesion molecule; LECAM-I; cDNA; (Rat)
A rat cDNA clone encoding an adhesion molecule, LECAM-1, has been isolated from the SD rat and the partial nucleotide sequence was determined. It encodes a peptide of 372 amino acids (aa), including a signal peptide of 38 aa. The protein has three tandem domains: a lectin domain, an EGF-like domain and two repeats of complement regulatory proteins (CR domain). The lectin binding domain has 93.2% and 81.4% and the EGF-like domain has 85.3% and 76.5% aa identity with those of mouse and human, respectively. In the CR repeat domain, the amino acid identity was 72.6% between human and rat and 71.8% between mouse and rat. Northern blot analysis detects the main transcript of about 3 kb in peripheral blood mononuclear cells (PBMC), spleen and thymus. The expression was down-regulated by mitogen stimulation of PBMC and spleen T cells. The protein encoded by this cDNA interacted with PPME when it was expressed on gp90 MELq4 negative mouse EL-4 cells.
LECAM-1 (mouse MEL-14 antigen and human LAM-1) was first reported as lymphocyte homing receptor that mediates lymph node specific migration of lymphocytes [1-5]. Molecular cloning of the c D N A of this protein revealed that LECAM-1 is one of the members of L E C A M or selectin family that has a common characteristic domain structure consisting of a lectin binding domain, an EGF-like domain and repeat units comforming to the homologous repeat structures of complement regulatory proteins (CR domain) [6]. Since this molecule has been shown to be expressed on a majority of leukocytes [7] and to mediate the localization of P M N to the site of inflammation [8], the way in which it directs lymph node specific migration of lymphocytes remains to be elucidated. In order to characterize the detailed functions of LECAM-1, we have isolated a c D N A that encodes the rat LECAM-1. The Agtl0 c D N A libraries were constructed from poly(A) + R N A of PBMC and thymus of SD rat according to the method of Gobler and Hoffman [9] with minor modifications. These libraries were screened by
Correspondence to: T. Watanabe, Department of Pathology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan. The sequence data in this paper have been submitted to the EMBL/GenBank and DDBJ Nucleotide Sequence Data bases under the accession number D10831.
plaque hybridization with 32p-labelled rat e D N A that was amplified by PCR from rat PBMC with a primer pair for mouse LECAM-1. The PCR primer that successfully amplified the rat c D N A sequence was as follows (the nucleotide position (n.p.) is that of mouse LECAM-1 [1]); 5' primer 5 ' - A C T C A C T G T T G G A C T T A C C A T - 3 ' (n.p. 160 to 180), 3' primer 5 ' - A C C A T G A C G G C T A C A G G A A T G - 3 ' (n.p. 1079-1059). Many positive clones were identified in each library. After plaque purification, the 2.9 kb e D N A insert was subcloned into pBluescriptlI (Stratagene). The partial nucleotide sequence was determined by the use of the dideoxy chain t e r m i n a t i o n m e t h o d [10] with Sequenase T M (USB). In the 3' untranslated region, the putative polyadenylation consensus signal, A A T A A A , was located 14 nucleotides from the start of poly(A) tail. The nucleotide sequence of 1272 bp that covers from the 5' end of the c D N A clone to the stop codon of the longest open reading frame and the derived amino acid sequence are shown in Fig. 1. This 1116 bp open reading frame encodes a protein of 372 amino acids. As the translation initiation site, the most 5' A T G codon conformed to the consensus sequence for optimal initiation ( A / G ) C C A T G [11] and was followed by a hydrophobic region of 38 amino acids that corresponds to signal peptide. This c D N A encodes a second hydrophobic region between amino acids 334 and 355 from the first methionine that may be a trans-mem-
322 C CTGAGTGCAGCACAC
C C TC C TTGTGCAAGAGCACAGCGCCC
CAGGT M
GCC
CGCAGAAGGACTTACAGAAGAGACC
S
A
Q
R
G
S
GAGTGCTCAGCGGGGC
V
F
P
CAGCAAGCCATGGTGTTT
W
S
F
L
K
TC CTGGAGCTTCC
L
W
GCAGAAGAC W
TGAG
R
C
Q
I
R
T
L
L
-31 120
CCATGGAGATGTCA
TGAAGCTGTGGATCAGGACAC
6O
C
C
-Ii 180
TGCTGTGTTG
+i D L L P H H TGATCTCCTGCCACACCATGGAAC N
W
E
GAACT
N
A
GGGAAAAT
I
G
I
K
E
I
R
K
I
V
E
E
I
K
R
CAAAC G
N
W
Y
K
A
Q
I
K
H
A
L
V
E
T
V
T
T
W
C
Q
Y
v
T
E
P
N
G
N
N
C
I
H
E
G
L
L
R
D
T
N
S
N
Q
G
TCTG
A
S
C TCC N
T
CAACAATAACACC
G
I~
T C CTAC
T
N
C
E
K
D
F
S
S
M
A
I
Q
K
K
Y Y CTACTAC
50 360
T
W
N
C
Q
TGC
CAACCAGAAT
C
I
L
E
T
D
C
E
T G
D
A
GAC
GAT
GC CTGTCA
S
C
N
CTT
C
D
P
TGT
GAC
CCAGGGTATTAT
G
Y
L
A
P
E
L
G
H
R
GCAACC
H
T
Q
S
Q
C
A
F
~
S
E
E
L
L
GAG
C TACT
P
I
C
G
N
A
TGGGAAT Q
K
T
E
C
GCAAAAACAGAGT
V
I
Q
C
G
A
P
L
A
G
150 660
M
M
E
C
S
H
P
L
A
~
F
S
F
T
c
S
S
G
~
W
T
C
S
W
S
C T GGT G
E
E
T
S
C TAG D
P
S
F
I
P
TCC TAGT
Y
N
L
I
T GACTTAAT
P
AGGCGACTATAACC A
D
CAGAAGAAAC
I
C
C CAATAT L
F
G
E
R
K
T
T GGGGAGAGAAAAACT Q
K
T
K
R
V
A
P
D
L
I
P
V
A
V
M
S
A
G
C
T
F
T
W
GGCATTTATCATTTGG
R
S
S
F
S
S CATC
K
I
G TGGC K
S TC E
L
A
R
C TGGCAAGGCGG
R
L
K
K
G
K
....
250
T
A
F
S
G
K
270 1020
290 1080
T T CT CAAAGATCAAGGA L
310 1140
S
Q
C TAAAAAAAGGCAAGAAATCGCAGGAAAGGAT
D D P Y * GGATGATCCTTACTGATTGATCCTTTGTGAGAGGAAAGTCATAAGGTACTAACAACCGCA
CCAAAACATTGG
C
CCCTCTTCATTCCAGTAGCTGTCATGGTCACTGCATTCTCAGGGCT I
230
960
AGAT
V
210
90O
GT C TGC
GT CAAAAGACAAAGAGAAGT
190
840
GGAAACTGGAC
CACTATGGAATGCAGTCACCCCCTCGCCAACTTCAGTTTTACTTCTGCTTGTACCTTCAC
C TGCT
170 720
ATATCTAGAGCCAATCTGTCAAGTGATTCAGTGCATGCCTCTAGCAGCACCGGATCTGGG
T
130 600
780
GT GGAGCATCT M
ii0
GG
CC C TGAGCTGGGTACCAT F
90 480
540
GC CA Y
70 420
D
C
K
TG
CCTCAC
TGCATC P
W
CTAAGGAAGAC
GGAAGTGGAAT
P
K
30 300
K
S
i0 240
T GC TATACA
CCCTTGGGAGACTTCAGCTTCCAGTCACAGTGTGCTTTCAACTGTTC
TGAGGGAAGC Y
P
V
TAGT
R
CAACAAAAC
GCCCCAGTGTCAGTATGTGATCCAATGTGAGCCTTTGAAGGC
GAACTGTATTCAC
L
K ~ C CAAAAAC
GTGGACATGGGTGGGAAC G
Y
I
W
D
T GGGGAGCCCAACAACAAG*~AGT
E
I TAT
ACAGATT
K
TAC
C
T
TAC
P
GAAAAC
R
Y
E K T L T TGGAAAAGACATTAC
TC T CT GC TACACAGC
TGTGGAAAC
N
CACAAT
L
G
K
GCAGC
C
T G GAGAATG P
C
T C TGCAAG
G
T GGGG
H C W T Y H Y S E TCACTGTTGGACTTACCATTATTCTGAAAGATCCAT
C TATATCAAGAGGGAACGAGAC
GAAAG E
F
CGG
CAAAGAAGCAGAGAAC
C GTGGAGAT
K
E Y T GAGTAT
CAGGAAAAT A
T
GCTAGAAAGT
N K R E AAATAAGAGAGAAAT
GATAGGAAT
R
G
E
R
M
330 1200 334 1260
1272
323 A) Lectin
domain
Rat
LECAM-I
WTYHYSERSMNWENARKFCKHNYTDLVAIQNKREIEYLEKTLPKNPTYYWIGIRKIGKTWTWVGTNKTL
Mouse
LECAM-I
*.°°,,,KP
Human
LECAM-I
° -°°-ooKp°
....
o-°°,,,Q°°°°°°°°,°°,,
• -QR,,R,
.....
Rat
LECAM-I
Mouse
LECAM-I
TKEAENWGTGEPNNKKSKEDCVEIYIKRERDSGKWNDDACHKRKAALCY .°ooooooAooooooooooooooooooooooooooooooooooooooo°
Human
LECAM-I
°m° °°°°°D°°°,-°°N°°°°,,,,°,,NK°A°°o,o,,°,°L°o°°°,
B) EGF-like
Rat
LECAM-I
CQPESCNRHGECVET
LECAM-I
• * °Go • -GR°oo,,,,,oH-
Human
LECAM-I
• °°W°
(1)CRI
.....
oM°o*°.
°SG°
INNNTC
ICDPGYYGPQCQY --,,A,,°,...,°
• • 0, , I - ° - Y , o N ° , V - - ° , ° , ° ° F
domain
domain
Rat
LECAM-I
VIQCEPLKAPELGTMNCIHPLGDFSFQSQCAFNCSEGSELLGNAKTECGASGNWTYLEPICQ
Mouse
LECAM-I
Human
LECAM-I
oVoooooEoooooooDooooeoNoooooKoooooo°oRooooToEoQoooooooSSPooo°o o°oooooEoooooooDoToSo oNoo oSo*oooSooo oTNoToIEEoTo opFo° oSSPo oT, o
(2)CR2
.....
domain
Mouse
C)CR
N-°°oS°Yo,°°°
°RD°°°°,,°,,°°A°,°°°°°°,°FSRS,,°.°oo°°°GI°°,°,°°°S.
domain
Rat Mouse
LECAM-I LECAM-I
Vl QCMPLAAPDLGTMECSHPLANFS FTSACTFTCSEETDLIGERKTVCRSSGSWS oVo oEo oEo*EooooDoIo • oGoo.oQoKoAoN, ooGREoLoTAEoQoGAo
Human
LECAM-I
• °ooE,
°S--..°IoNooooooSo.oo.ooooI...GoEo°oKK.
S pSp I CQ oNooooEo, o.
°IoEo,°I.
°Nooooo°
Fig. 2. Alignment of amino acid sequences of rat, mouse and human LECAMI. Amino acid sequences of three domains (lectin domain, EGF-like domain and CR domain) are aligned in single-letter abbreviation for amino acids. The dots represent an amino acid that is identical to the residue of rat L E C A M - h
brane region. The sequence homology to human and mouse counterparts at the nucleotide level was 79.9% and 86.2%, respectively. Homologies of the deduced amino acid sequence to those of human and rat LECAM-1 were as follows; in the lectin binding domain (amino acids +1-118), the amino acids were identical in 110 out of 118 (93.2% identity) between rat and mouse, and in 96 out of 118 (81.4% identity) between rat and human. In the EGF-like domain (amino acids 122-155), they were identical in 29 out of 34 (85.3% identity) between rat and mouse and 26 out of 34 (76.5% identity) between rat and human. In the CR domains (amino acids 156-279), they were identical in 89 out of 124 (71.8%) between rat and mouse, and in 90 out of 124 (72.6%) between rat and human. Amino acid sequence of rat CR2 showed higher homology to that of human (48 out of 62 identity, 77.4%) than to mouse (39 out of 62 identity, 62.9%). The
comparison of the amino acid sequence of three domains of rat LECAM-1 with those of the mouse and the human is shown in Fig. 2. Northern blot analysis with a cDNA insert as a probe detected a major transcript of about 3 kb and a minor one of 1.9 kb in PBMC, spleen, thymus and lung, but not in liver, brain or testis (Fig. 3). The expression of rat LECAM-1 was mainly restricted to lymphoid cells and tissues, and the signals detected in the lung were possibly due to the intravascular leukocyte pool in this organ. We could not confirm the expression of smaller transcripts in the testis reported by Siegelman et al. [1]. The expression of LECAM-1 in PBMCs was down-regulated after T cell activation by PHA-P (0.1%) plus PMA (20/xg/ml), or ConA ( 2 0 / ~ g / m l ) for 18 h. Previous work has demonstrated that human and mouse LECAM-1 react with yeast cell carbohydrate PPME in a specific manner [12]. In order to test for
Fig. 1. The nucleotide and amino acid sequences of the rat LECAM1. Partial nucleotide sequence that covers from 5' end of the cDNA to the stop codon of the open reading frame is shown with deduced amino acid sequence in single-letter abbreviation for amino acids. The initial tryptophan residue of presumed mature protein is indicated by + 1. The putative transmembrane sequence is underlined. Potential N-linked glycosylation sites are indicated by double-underline.
324 carbohydrate-binding ability of the rat protein encoded by this cDNA, the interaction with fluoresceinated PPME of EL-4 ceils expressing the rat LECAM-1 cDNA was analyzed by cytofluorometry. Murine lymphoma EL-4 cells that are negative for endogenous LECAM-1 expression were transfected with BCMGSNeo vector [13] containing rat LECAM-1 cDNA by electroporation [14] and the transformed EL-4 cells were selected by G418 at the concentration of 400 /xg/ml. After cloning by limiting dilution, they were tested for the expression of rat LECAM-1 mRNA and the ability of interaction with PPME. As shown in Fig. 4, only EL-4 cells expressing rat LECAM-1 mRNA (RM-1) were intensely stained with fluoresceinated PPME, whereas untransfected EL-4 cells or EL-4 ceils transfected with rat LECAM-1 cDNA but lost its expression (RM-2) bound PPME only marginally. Procurement of a cDNA encoding the rat LECAM-1 will facilitate investigation on the physiological significance of LECAM-1 in lymphocyte recirculation and also localization of leukocytes to the site of inflammation by the use of rats. We have produced a soluble form of rat LECAM-1 utilizing this cDNA and are
A)
1 2 3456
A
1234
!
B
/l
iA
a
¢1
b
e
e
f
Fig. 4. Interaction of rat LECAMI with PPME. (A) Northern blot analysis of the expression of rat LECAMI introduced into EL-4 cells. 1 /zg of poly(A) + selected RNA was applied on each lane. Lane 1, rat spleen; lane 2, untransfected EL-4 cell; lane 3, RM-1 cell; lane 4, RM-2 cell. RM-1 and RM-2 cells are EL-4 cells that were transfected with rat LECAM1 expression vector and cloned by limiting dilution under the G418 selection. (B) Cytofluorometric analysis of interaction of PPME. a, b, c, negative control treated with buffer alone; d, e, f, treated with 10 /xg/ml of PPME-FITC for 40 min. a and d, EL-4 cells; b and e, RM-2 cells; c and f. RM-1 cells.
using it as immunogen to produce monoclonal antibodies against rat LECAM-1 and also to identify ligands for LECAM-1 in this animal species (T. Tamatani, K. Kuita, T. Watanabe and M. Miyasaka, unpublished data). References
B)
123
Fig. 3. Northern blot analysis of the expression of LECAM1 in rat tissues and PBMC. (A) Expression in rat tissues. 1 ,ag of poly(A) + selected RNA from each tissue was analyzed. Lane 1, spleen; lane 2, thymus; lane 3, liver; lane 4, lung; lane 5, brain; lane 6, testis. (B) Down regulation by mitogen stimulation. PBMC was stimulated by PHA (0.1%) plus PMA (20 ng/ml) or ConA (20 /xg/ml) for 18 h. 10p~g of total RNA from each sample was analyzed. Lane 1, fresh PBMC; lane 2, PBMC stimulated by ConA; lane 3, PBMC stimulated by PHA and PMA. Arrowheads indicate the position of 28S and 18S rRNA.
1 Siegelman, M.H., Van de Rijn, M. and Weissman, I.L. (1989) Science 243, 1165-1172. 2 Siegelman, M.H. and Weissman, I.L. (1989) Proc. Natl. Acad. Sci. USA 86, 5562-5566. 3 Lasky, L.A., Singer, M.S., Yednock, T.A., Dowbenko, D., Fennie, C., Rodriguez, H., Nguyen, T., Stachel and Rosen, S.D. (1989) Cell 56, 1045-1055. 4 Tedder, T.F., Isaacs, G.M., Ernst, T.J., Demetri, G.D., Adler, D.A. and Disteche, C.M. (1989) J. Exp. Med. 170, 123-133. 5 Camerini, D., James, S.R, Stamenkovic, I. and Seed, B. (1989) Nature 342, 78-82. 6 Brandley, B.K,, Swiedler, S.J. and Robbins, P.W. (19901 Cell 63, 861-863. 7 Stoolman, L.M. (1989) Cell 56, 907-910. 8 Jutila, M.A., Rott, L., Berg, E.L. and Butcher, E.C. (1989) J. Immunol. 143, 3318-3324. 9 Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263-269. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (19771 Proc. Natl. Acad. Sci. USA 74, 5463-5467. 11 Kozak, M. (19861 Cell 44, 283-292. 12 Yednock, T.A., Stoolman, L.M. and Rosen, S.D. (1987) J. Cell. Biol. 104, 713-723. 13 Karasuyama, H. and Melchers, F. (1988) Eur. J. Immunol. 18, 97 104. 14 Potter, H., Weir, L. and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161-7165.
Biochimica et Biophysica Acta, 1131 (1992) 321-324 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90374
Short Sequence-Paper
Sequence and expression of a rat c D N A for LECAM-1 Toshiki Watanabe a , b Yuru Song ", Youko Hirayama c, Takuya Tamatani Keisuke Kuida b and Masayuki Miyasaka b
b
a Department of Pathology, Institute of Medical Science, The University of Tokyo, b Department of Immunology, Tokyo Metropolitan Institute for Medical Science and c Department of Viral Oncology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo (Japan) (Received 14 April 1992)
Key words: Adhesion molecule; LECAM-I; cDNA; (Rat)
A rat cDNA clone encoding an adhesion molecule, LECAM-1, has been isolated from the SD rat and the partial nucleotide sequence was determined. It encodes a peptide of 372 amino acids (aa), including a signal peptide of 38 aa. The protein has three tandem domains: a lectin domain, an EGF-like domain and two repeats of complement regulatory proteins (CR domain). The lectin binding domain has 93.2% and 81.4% and the EGF-like domain has 85.3% and 76.5% aa identity with those of mouse and human, respectively. In the CR repeat domain, the amino acid identity was 72.6% between human and rat and 71.8% between mouse and rat. Northern blot analysis detects the main transcript of about 3 kb in peripheral blood mononuclear cells (PBMC), spleen and thymus. The expression was down-regulated by mitogen stimulation of PBMC and spleen T cells. The protein encoded by this cDNA interacted with PPME when it was expressed on gp90 MELq4 negative mouse EL-4 cells.
LECAM-1 (mouse MEL-14 antigen and human LAM-1) was first reported as lymphocyte homing receptor that mediates lymph node specific migration of lymphocytes [1-5]. Molecular cloning of the c D N A of this protein revealed that LECAM-1 is one of the members of L E C A M or selectin family that has a common characteristic domain structure consisting of a lectin binding domain, an EGF-like domain and repeat units comforming to the homologous repeat structures of complement regulatory proteins (CR domain) [6]. Since this molecule has been shown to be expressed on a majority of leukocytes [7] and to mediate the localization of P M N to the site of inflammation [8], the way in which it directs lymph node specific migration of lymphocytes remains to be elucidated. In order to characterize the detailed functions of LECAM-1, we have isolated a c D N A that encodes the rat LECAM-1. The Agtl0 c D N A libraries were constructed from poly(A) + R N A of PBMC and thymus of SD rat according to the method of Gobler and Hoffman [9] with minor modifications. These libraries were screened by
Correspondence to: T. Watanabe, Department of Pathology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan. The sequence data in this paper have been submitted to the EMBL/GenBank and DDBJ Nucleotide Sequence Data bases under the accession number D10831.
plaque hybridization with 32p-labelled rat e D N A that was amplified by PCR from rat PBMC with a primer pair for mouse LECAM-1. The PCR primer that successfully amplified the rat c D N A sequence was as follows (the nucleotide position (n.p.) is that of mouse LECAM-1 [1]); 5' primer 5 ' - A C T C A C T G T T G G A C T T A C C A T - 3 ' (n.p. 160 to 180), 3' primer 5 ' - A C C A T G A C G G C T A C A G G A A T G - 3 ' (n.p. 1079-1059). Many positive clones were identified in each library. After plaque purification, the 2.9 kb e D N A insert was subcloned into pBluescriptlI (Stratagene). The partial nucleotide sequence was determined by the use of the dideoxy chain t e r m i n a t i o n m e t h o d [10] with Sequenase T M (USB). In the 3' untranslated region, the putative polyadenylation consensus signal, A A T A A A , was located 14 nucleotides from the start of poly(A) tail. The nucleotide sequence of 1272 bp that covers from the 5' end of the c D N A clone to the stop codon of the longest open reading frame and the derived amino acid sequence are shown in Fig. 1. This 1116 bp open reading frame encodes a protein of 372 amino acids. As the translation initiation site, the most 5' A T G codon conformed to the consensus sequence for optimal initiation ( A / G ) C C A T G [11] and was followed by a hydrophobic region of 38 amino acids that corresponds to signal peptide. This c D N A encodes a second hydrophobic region between amino acids 334 and 355 from the first methionine that may be a trans-mem-
322 C CTGAGTGCAGCACAC
C C TC C TTGTGCAAGAGCACAGCGCCC
CAGGT M
GCC
CGCAGAAGGACTTACAGAAGAGACC
S
A
Q
R
G
S
GAGTGCTCAGCGGGGC
V
F
P
CAGCAAGCCATGGTGTTT
W
S
F
L
K
TC CTGGAGCTTCC
L
W
GCAGAAGAC W
TGAG
R
C
Q
I
R
T
L
L
-31 120
CCATGGAGATGTCA
TGAAGCTGTGGATCAGGACAC
6O
C
C
-Ii 180
TGCTGTGTTG
+i D L L P H H TGATCTCCTGCCACACCATGGAAC N
W
E
GAACT
N
A
GGGAAAAT
I
G
I
K
E
I
R
K
I
V
E
E
I
K
R
CAAAC G
N
W
Y
K
A
Q
I
K
H
A
L
V
E
T
V
T
T
W
C
Q
Y
v
T
E
P
N
G
N
N
C
I
H
E
G
L
L
R
D
T
N
S
N
Q
G
TCTG
A
S
C TCC N
T
CAACAATAACACC
G
I~
T C CTAC
T
N
C
E
K
D
F
S
S
M
A
I
Q
K
K
Y Y CTACTAC
50 360
T
W
N
C
Q
TGC
CAACCAGAAT
C
I
L
E
T
D
C
E
T G
D
A
GAC
GAT
GC CTGTCA
S
C
N
CTT
C
D
P
TGT
GAC
CCAGGGTATTAT
G
Y
L
A
P
E
L
G
H
R
GCAACC
H
T
Q
S
Q
C
A
F
~
S
E
E
L
L
GAG
C TACT
P
I
C
G
N
A
TGGGAAT Q
K
T
E
C
GCAAAAACAGAGT
V
I
Q
C
G
A
P
L
A
G
150 660
M
M
E
C
S
H
P
L
A
~
F
S
F
T
c
S
S
G
~
W
T
C
S
W
S
C T GGT G
E
E
T
S
C TAG D
P
S
F
I
P
TCC TAGT
Y
N
L
I
T GACTTAAT
P
AGGCGACTATAACC A
D
CAGAAGAAAC
I
C
C CAATAT L
F
G
E
R
K
T
T GGGGAGAGAAAAACT Q
K
T
K
R
V
A
P
D
L
I
P
V
A
V
M
S
A
G
C
T
F
T
W
GGCATTTATCATTTGG
R
S
S
F
S
S CATC
K
I
G TGGC K
S TC E
L
A
R
C TGGCAAGGCGG
R
L
K
K
G
K
....
250
T
A
F
S
G
K
270 1020
290 1080
T T CT CAAAGATCAAGGA L
310 1140
S
Q
C TAAAAAAAGGCAAGAAATCGCAGGAAAGGAT
D D P Y * GGATGATCCTTACTGATTGATCCTTTGTGAGAGGAAAGTCATAAGGTACTAACAACCGCA
CCAAAACATTGG
C
CCCTCTTCATTCCAGTAGCTGTCATGGTCACTGCATTCTCAGGGCT I
230
960
AGAT
V
210
90O
GT C TGC
GT CAAAAGACAAAGAGAAGT
190
840
GGAAACTGGAC
CACTATGGAATGCAGTCACCCCCTCGCCAACTTCAGTTTTACTTCTGCTTGTACCTTCAC
C TGCT
170 720
ATATCTAGAGCCAATCTGTCAAGTGATTCAGTGCATGCCTCTAGCAGCACCGGATCTGGG
T
130 600
780
GT GGAGCATCT M
ii0
GG
CC C TGAGCTGGGTACCAT F
90 480
540
GC CA Y
70 420
D
C
K
TG
CCTCAC
TGCATC P
W
CTAAGGAAGAC
GGAAGTGGAAT
P
K
30 300
K
S
i0 240
T GC TATACA
CCCTTGGGAGACTTCAGCTTCCAGTCACAGTGTGCTTTCAACTGTTC
TGAGGGAAGC Y
P
V
TAGT
R
CAACAAAAC
GCCCCAGTGTCAGTATGTGATCCAATGTGAGCCTTTGAAGGC
GAACTGTATTCAC
L
K ~ C CAAAAAC
GTGGACATGGGTGGGAAC G
Y
I
W
D
T GGGGAGCCCAACAACAAG*~AGT
E
I TAT
ACAGATT
K
TAC
C
T
TAC
P
GAAAAC
R
Y
E K T L T TGGAAAAGACATTAC
TC T CT GC TACACAGC
TGTGGAAAC
N
CACAAT
L
G
K
GCAGC
C
T G GAGAATG P
C
T C TGCAAG
G
T GGGG
H C W T Y H Y S E TCACTGTTGGACTTACCATTATTCTGAAAGATCCAT
C TATATCAAGAGGGAACGAGAC
GAAAG E
F
CGG
CAAAGAAGCAGAGAAC
C GTGGAGAT
K
E Y T GAGTAT
CAGGAAAAT A
T
GCTAGAAAGT
N K R E AAATAAGAGAGAAAT
GATAGGAAT
R
G
E
R
M
330 1200 334 1260
1272
323 A) Lectin
domain
Rat
LECAM-I
WTYHYSERSMNWENARKFCKHNYTDLVAIQNKREIEYLEKTLPKNPTYYWIGIRKIGKTWTWVGTNKTL
Mouse
LECAM-I
*.°°,,,KP
Human
LECAM-I
° -°°-ooKp°
....
o-°°,,,Q°°°°°°°°,°°,,
• -QR,,R,
.....
Rat
LECAM-I
Mouse
LECAM-I
TKEAENWGTGEPNNKKSKEDCVEIYIKRERDSGKWNDDACHKRKAALCY .°ooooooAooooooooooooooooooooooooooooooooooooooo°
Human
LECAM-I
°m° °°°°°D°°°,-°°N°°°°,,,,°,,NK°A°°o,o,,°,°L°o°°°,
B) EGF-like
Rat
LECAM-I
CQPESCNRHGECVET
LECAM-I
• * °Go • -GR°oo,,,,,oH-
Human
LECAM-I
• °°W°
(1)CRI
.....
oM°o*°.
°SG°
INNNTC
ICDPGYYGPQCQY --,,A,,°,...,°
• • 0, , I - ° - Y , o N ° , V - - ° , ° , ° ° F
domain
domain
Rat
LECAM-I
VIQCEPLKAPELGTMNCIHPLGDFSFQSQCAFNCSEGSELLGNAKTECGASGNWTYLEPICQ
Mouse
LECAM-I
Human
LECAM-I
oVoooooEoooooooDooooeoNoooooKoooooo°oRooooToEoQoooooooSSPooo°o o°oooooEoooooooDoToSo oNoo oSo*oooSooo oTNoToIEEoTo opFo° oSSPo oT, o
(2)CR2
.....
domain
Mouse
C)CR
N-°°oS°Yo,°°°
°RD°°°°,,°,,°°A°,°°°°°°,°FSRS,,°.°oo°°°GI°°,°,°°°S.
domain
Rat Mouse
LECAM-I LECAM-I
Vl QCMPLAAPDLGTMECSHPLANFS FTSACTFTCSEETDLIGERKTVCRSSGSWS oVo oEo oEo*EooooDoIo • oGoo.oQoKoAoN, ooGREoLoTAEoQoGAo
Human
LECAM-I
• °ooE,
°S--..°IoNooooooSo.oo.ooooI...GoEo°oKK.
S pSp I CQ oNooooEo, o.
°IoEo,°I.
°Nooooo°
Fig. 2. Alignment of amino acid sequences of rat, mouse and human LECAMI. Amino acid sequences of three domains (lectin domain, EGF-like domain and CR domain) are aligned in single-letter abbreviation for amino acids. The dots represent an amino acid that is identical to the residue of rat L E C A M - h
brane region. The sequence homology to human and mouse counterparts at the nucleotide level was 79.9% and 86.2%, respectively. Homologies of the deduced amino acid sequence to those of human and rat LECAM-1 were as follows; in the lectin binding domain (amino acids +1-118), the amino acids were identical in 110 out of 118 (93.2% identity) between rat and mouse, and in 96 out of 118 (81.4% identity) between rat and human. In the EGF-like domain (amino acids 122-155), they were identical in 29 out of 34 (85.3% identity) between rat and mouse and 26 out of 34 (76.5% identity) between rat and human. In the CR domains (amino acids 156-279), they were identical in 89 out of 124 (71.8%) between rat and mouse, and in 90 out of 124 (72.6%) between rat and human. Amino acid sequence of rat CR2 showed higher homology to that of human (48 out of 62 identity, 77.4%) than to mouse (39 out of 62 identity, 62.9%). The
comparison of the amino acid sequence of three domains of rat LECAM-1 with those of the mouse and the human is shown in Fig. 2. Northern blot analysis with a cDNA insert as a probe detected a major transcript of about 3 kb and a minor one of 1.9 kb in PBMC, spleen, thymus and lung, but not in liver, brain or testis (Fig. 3). The expression of rat LECAM-1 was mainly restricted to lymphoid cells and tissues, and the signals detected in the lung were possibly due to the intravascular leukocyte pool in this organ. We could not confirm the expression of smaller transcripts in the testis reported by Siegelman et al. [1]. The expression of LECAM-1 in PBMCs was down-regulated after T cell activation by PHA-P (0.1%) plus PMA (20/xg/ml), or ConA ( 2 0 / ~ g / m l ) for 18 h. Previous work has demonstrated that human and mouse LECAM-1 react with yeast cell carbohydrate PPME in a specific manner [12]. In order to test for
Fig. 1. The nucleotide and amino acid sequences of the rat LECAM1. Partial nucleotide sequence that covers from 5' end of the cDNA to the stop codon of the open reading frame is shown with deduced amino acid sequence in single-letter abbreviation for amino acids. The initial tryptophan residue of presumed mature protein is indicated by + 1. The putative transmembrane sequence is underlined. Potential N-linked glycosylation sites are indicated by double-underline.
324 carbohydrate-binding ability of the rat protein encoded by this cDNA, the interaction with fluoresceinated PPME of EL-4 ceils expressing the rat LECAM-1 cDNA was analyzed by cytofluorometry. Murine lymphoma EL-4 cells that are negative for endogenous LECAM-1 expression were transfected with BCMGSNeo vector [13] containing rat LECAM-1 cDNA by electroporation [14] and the transformed EL-4 cells were selected by G418 at the concentration of 400 /xg/ml. After cloning by limiting dilution, they were tested for the expression of rat LECAM-1 mRNA and the ability of interaction with PPME. As shown in Fig. 4, only EL-4 cells expressing rat LECAM-1 mRNA (RM-1) were intensely stained with fluoresceinated PPME, whereas untransfected EL-4 cells or EL-4 ceils transfected with rat LECAM-1 cDNA but lost its expression (RM-2) bound PPME only marginally. Procurement of a cDNA encoding the rat LECAM-1 will facilitate investigation on the physiological significance of LECAM-1 in lymphocyte recirculation and also localization of leukocytes to the site of inflammation by the use of rats. We have produced a soluble form of rat LECAM-1 utilizing this cDNA and are
A)
1 2 3456
A
1234
!
B
/l
iA
a
¢1
b
e
e
f
Fig. 4. Interaction of rat LECAMI with PPME. (A) Northern blot analysis of the expression of rat LECAMI introduced into EL-4 cells. 1 /zg of poly(A) + selected RNA was applied on each lane. Lane 1, rat spleen; lane 2, untransfected EL-4 cell; lane 3, RM-1 cell; lane 4, RM-2 cell. RM-1 and RM-2 cells are EL-4 cells that were transfected with rat LECAM1 expression vector and cloned by limiting dilution under the G418 selection. (B) Cytofluorometric analysis of interaction of PPME. a, b, c, negative control treated with buffer alone; d, e, f, treated with 10 /xg/ml of PPME-FITC for 40 min. a and d, EL-4 cells; b and e, RM-2 cells; c and f. RM-1 cells.
using it as immunogen to produce monoclonal antibodies against rat LECAM-1 and also to identify ligands for LECAM-1 in this animal species (T. Tamatani, K. Kuita, T. Watanabe and M. Miyasaka, unpublished data). References
B)
123
Fig. 3. Northern blot analysis of the expression of LECAM1 in rat tissues and PBMC. (A) Expression in rat tissues. 1 ,ag of poly(A) + selected RNA from each tissue was analyzed. Lane 1, spleen; lane 2, thymus; lane 3, liver; lane 4, lung; lane 5, brain; lane 6, testis. (B) Down regulation by mitogen stimulation. PBMC was stimulated by PHA (0.1%) plus PMA (20 ng/ml) or ConA (20 /xg/ml) for 18 h. 10p~g of total RNA from each sample was analyzed. Lane 1, fresh PBMC; lane 2, PBMC stimulated by ConA; lane 3, PBMC stimulated by PHA and PMA. Arrowheads indicate the position of 28S and 18S rRNA.
1 Siegelman, M.H., Van de Rijn, M. and Weissman, I.L. (1989) Science 243, 1165-1172. 2 Siegelman, M.H. and Weissman, I.L. (1989) Proc. Natl. Acad. Sci. USA 86, 5562-5566. 3 Lasky, L.A., Singer, M.S., Yednock, T.A., Dowbenko, D., Fennie, C., Rodriguez, H., Nguyen, T., Stachel and Rosen, S.D. (1989) Cell 56, 1045-1055. 4 Tedder, T.F., Isaacs, G.M., Ernst, T.J., Demetri, G.D., Adler, D.A. and Disteche, C.M. (1989) J. Exp. Med. 170, 123-133. 5 Camerini, D., James, S.R, Stamenkovic, I. and Seed, B. (1989) Nature 342, 78-82. 6 Brandley, B.K,, Swiedler, S.J. and Robbins, P.W. (19901 Cell 63, 861-863. 7 Stoolman, L.M. (1989) Cell 56, 907-910. 8 Jutila, M.A., Rott, L., Berg, E.L. and Butcher, E.C. (1989) J. Immunol. 143, 3318-3324. 9 Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263-269. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (19771 Proc. Natl. Acad. Sci. USA 74, 5463-5467. 11 Kozak, M. (19861 Cell 44, 283-292. 12 Yednock, T.A., Stoolman, L.M. and Rosen, S.D. (1987) J. Cell. Biol. 104, 713-723. 13 Karasuyama, H. and Melchers, F. (1988) Eur. J. Immunol. 18, 97 104. 14 Potter, H., Weir, L. and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161-7165.
