Cloning of bovine CD69

Article (PDF Available)inVeterinary Immunology and Immunopathology 88(1-2):43-8 · October 2002with7 Reads
DOI: 10.1016/S0165-2427(02)00125-3 · Source: PubMed
Abstract
CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen. Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that, bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-length cDNA contained an 80bp 5' untranslated region, followed by a 600bp coding region and AU-rich motifs in a 3' untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and 78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation of lymphocytes.
Cloning of bovine CD69
J.S. Ahn
a
, M.J. Hamilton
a
, W.C. Davis
a
, Y.H. Park
b,*
a
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine,
Washington State University, Pullman, WA 99164-7040, USA
b
Department of Microbiology, School of Agricultural Biotechnology, College of Veterinary Medicine,
Seoul National University, Seo-Doon Dong 103, Kwon-Sun Gu, 441-744 Suwon, South Korea
Received 26 October 2001; received in revised form 18 March 2002; accepted 18 March 2002
Abstract
CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen.
Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that,
bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-
length cDNA contained an 80 bp 5
0
untranslated region, followed by a 600 bp coding region and AU-rich motifs in a 3
0
untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and
78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine
CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has
the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be
important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has
two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation
of lymphocytes. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: CD69; Cattle; Cloning; RACE; cDNA library
1. Introduction
The activation of T-lymphocytes results in the
expression of many genes, including CD69 (Ullman
et al., 1990). CD69 is one of the earliest activation
antigens, expressed upon stimulation of T-lympho-
cytes with cytokines, mitogens or via molecules on
the cell surface (Hara et al., 1986; Lanier et al., 1988;
Risso et al., 1989; Testi et al., 1989). It appears on
T-lymphocytes within 1–2 h, reaching a peak between
18 and 24 h, and is still readily detectable at 72 h
following stimulation. In contrast, the transcription of
CD69 reaches a peak within 30–60 min and drops to
nearly resting levels by 8 h post-stimulation (Testi
et al., 1989; Ziegler et al., 1994). This rapid down-
regulation is associated with AU-rich motifs in the
3
0
-end of CD69 (Lopez-Cabrera et al., 1993; Santis
et al., 1995). As resting lymphocytes do not express
CD69, the expression of CD69 has been used as a very
early activation marker of T-lymphocytes. In addition
to readily inducible expression on the surface cells
of most lymphoid cells, CD69 can be induced on
mouse macrophages, neutrophils, and eosinophils,
while it is constitutively expressed on CD3
bright
thy-
mocytes, human monocytes, platelets, and epidermal
Veterinary Immunology and Immunopathology 88 (2002) 43–48
Abbreviations: RACE, rapid amplification of cDNA ends
*
Corresponding author. Tel.: þ82-31-290-2735;
fax: þ82-31-295-7524.
E-mail address: yhp@snu.ac.kr (Y.H. Park).
0165-2427/02/$ see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0165-2427(02)00125-3
Langerhans cells (Marzio et al., 1999). Since CD69 is
expressed at an early stage of positive selection, it is a
useful marker to study the mechanisms of lymphocyte
maturation (Jung et al., 1990; Bendelac et al., 1992;
Swat et al., 1993). Moreover, studies of eosinophilic
pneumonia, asthma, HIV-1 infection, and dengue
hemorrhagic fever indicate that CD69 can be useful
in studying the pathogenesis of diseases (Nishikawa
et al., 1992; Hartnell et al., 1993; De Martino et al.,
1999; Green et al., 1999). Although CD69 is well
characterized in human and mouse, information on
bovine CD69 is not yet available. Thus, this study was
performed to clone and characterize bovine CD69.
2. Materials and methods
2.1. Isolation and culture of bovine peripheral
blood lymphocytes
Blood was collected from a Bos Taurus Holstein
by venipuncture of the jugular vein into 1/5 volume
acidcitratedextrose (ACD) and was centrifuged at
1500 rpm for 30 min. The buffy coat was harvested
and subjected to density gradient centrifugation using
Accupaque (Accurate Chemical, Westbury, NY) to
obtain peripheral blood lymphocytes. Since PMA is
a strong inducer of CD69, peripheral blood lympho-
cytes were stimulated with PMA (10 ng/ml) for 36h
in RPMI supplemented with 2 mM
L-glutamine and
13% bovine serum.
2.2. cDNA library construction
Total RNA and mRNA were isolated using Trizol
(GIBCO BRL, Rockville, MD) and FastTrack
TM
mRNA isolation kit (Invitrogen, Carlsbad, CA),
respectively. A cDNA library was constructed accord-
ing to the manufacturers protocol (Stratagene, La
Jolla, CA). In brief, 5 mg of mRNA was used to
synthesize the rst strand, and the second strand
was synthesized according to Gubler and Hoffmans
method (1983). Double strand cDNA was digested
with XhoI, following EcoRI adapter ligation and
fractionated on a 1% agarose gel. cDNA fraction
larger than 0.5 kbp was harvested using a gel extrac-
tion kit (Qiagen, Valencia, CA). One hundred nano-
gram of puried cDNA was ligated with 1 mg of ZAP
Express vector (Stratagene). The titer of the primary
cDNA library was 1 10
6
pfu. Primary cDNA library
was amplied (6:0 10
9
pfu/ml) and used in PCR to
clone bovine CD69.
2.3. PCR amplification of bovine CD69 through
RACE method
Attempts to amplify bovine CD69 failed using two
degenerative primers. RACE (rapid amplication of
cDNA ends) was applied to amplify bovine CD69 with
a highly consensus gene-specic primer and a ZAP
Express vector-specic primer. A gene-specic PCR
primer, BoCD69F 5
0
-GTG GGC CAATAC AAT TGT
CCA GG-3
0
was designed based on the conserved
amino acid sequence (VGQYNCPG) of human and
mouse CD69. It was used to amplify 3
0
-end of bovine
CD69 with a ZAP Express vector-specic primer,
ZAPR 5
0
-AAC GAC GGC CAG TGA ATT GT-3
0
.
The PCR reactions were performed in 100 ml volumes
containing 50 mM KCl, 10 mM TrisHCl, pH 8.8,
1.5 mM MgCl
2
, 100 mM of each dNTP, 15 pmol of
each primer, 5 ml of amplied cDNA library, and 2.5 U
of Taq Polymerase (GIBCO BRL). Reaction mixtures
were preheated at 94 8C for 5 min, followed by three
cycles of denaturing at 94 8C for 30 s, annealing at
53 8C for 30 s, and polymerization at 72 8C for 1 min.
The annealing temperature was then increased to
Fig. 1. Gel electrophoresis of DNA marker and PCR products.
Electrophoresis was performed using a 1% agarose gel in 1 TAE
buffer containing 0.5 mg/ml ethidium bromide. Lane 1, 2 kbp
marker (Research Genetics); lane 2, PCR amplication of the
3
0
-end of bovine CD69; lane 3, PCR amplication of the 5
0
-end of
bovine CD69.
44 J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 43–48
Fig. 2. Nucleotide sequence of bovine CD69 and deduced amino acid sequence. Potential glycosylation sites are marked with an
, and
potential rapid degradation signals are underlined.
J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 4348 45
58 8C for 30 cycles. PCR product was cloned into PCR
2.1 (Invitrogen) and sequenced using ABI 373
(Applied Biosystem, Bedford, MA). Based on the
sequencing data, a gene-specic primer, BoCD69R
5
0
-GCA TTT GCC CAC AGT TGT CAT A-3
0
, was
designed downstream of stop codon and was used to
amplify 5
0
-end of bovine CD69 with a ZAP Express
vector-specic primer, ZAPF 5
0
-TGA CCT TGA TTA
Fig. 3. Multiple sequence alignment of human, bovine, and mouse CD69s. Shaded residues represent identities among the sequences. Highly
conserved amino acid residues in C-type lectin family are marked with an
.
46 J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 4348
CGC CAA GC-3
0
. PCR was run for 30 cycles of
denaturing at 94 8C for 30 s, annealing at 58 8C for
30 s, polymerization at 72 8C for 30 s (Fig. 1). PCR
product was cloned into PCR 2.1 and sequenced.
3. Results and discussion
We attempted to clone a full-length bovine CD69
cDNA using RACE from an expression cDNA library
constructed following the stimulation with PMA for
36 h. Because amplication of bovine CD69 failed
using two degenerative primers, we constructed a
cDNA library. RACE was successful in amplifying
the 3
0
-end of bovine CD69. The length of the PCR
product was 1562 bp, including 118 bp of ZAP
Express vector sequences, an expected size based
on human CD69 (Hamann et al., 1993; Ziegler
et al., 1993; Lopez-Cabrera et al., 1993). Based on
the sequencing data of the 3
0
-end of bovine CD69,
a gene-specic primer was designed and used to
amplify 5
0
-end of bovine CD69 with another ZAP
Express-specic primer. The length of the PCR pro-
duct was 851 bp, including 100 bp of ZAP Express
vector (Fig. 1). The full length of bovine CD69 was
1710 bp (GenBank accession number AF272828;
Figs. 2 and 3). Bovine CD69 contained an 80 bp of
the 5
0
-end untranslated region, an open reading frame
(ORF) of 199 amino acids and potential AU-rich
motifs in the 3
0
-end. The levels of nucleotide sequence
identity of bovine CD69 with the human and mouse
CD69 were 78.2 and 69.4%, respectively. The identity
of deduced amino acids was slightly higher between
bovine and human CD69, 62.3%, compared with
bovine and mouse CD69, 56.3%, and human and
mouse CD69, 58%. Bovine CD69 contains highly
conserved amino acid sequences found in C-type
lectin family (Ziegler et al., 1993; Lopez-Cabrera
et al., 1993). The conserved residues may be important
for conformation and binding to the unidentied
ligand for bovine CD69. Human and mouse CD69
have three intra-chain disulde bonds, Cys
85
Cys
96
,
Cys
113
Cys
194
, and Cys
173
Cys
186
(Natarajan et al.,
2000; Llera et al., 2001). Interestingly, bovine CD69
as well as rat Kupffer cell receptor, a member of C-
type lectin family, do not have Cys
96
(Hamann et al.,
1993). Because Cys
85
precedes rst b0 strand and
Cys
96
is located within b1 strand at the N-terminus,
Tyr
96
does not change the structure of bovine CD69
signicantly (Natarajan et al., 2000; Llera et al.,
2001). Bovine CD69 contains two typical glycosyla-
tion sites (NXS/T; Hart et al., 1979) and one aty-
pical glycosylation site (NXC) in the extracellular
domain (Bause and Legler, 1981; Vance et al., 1997),
whereas mouse CD69 contains three typical N-linked
glycosylation sites. Human CD69 contains one typical
N-linked glycosylation site and one atypical glycosy-
lation site. In addition, bovine and mouse CD69
contain two casein kinase-2 (CK-2) phosphorylation
sites (S/TXXD/E) in the cytoplasmic tail (Pinna,
1990) while human CD69 contains two CK-2 phos-
phorylation sites and one protein kinase C (PKC)
phosphorylation site (S/TXK/R; Woodgett et al.,
1986). These observations suggest that bovine CD69
may be important for the activation of lymphocytes.
Acknowledgements
This work was supported in part by Grants from the
USDA-NRICGP no. 98-02-02480, WNV-00138 and
Brain Korea 21 project.
References
Bause, E., Legler, G., 1981. The role of the hydroxy amino acid in the
triplet sequence AsnXaaThr(Ser) for the N-glycosylation step
during glycoprotein biosynthesis. Biochem. J. 195, 639644.
Bendelac, A., Matzinger, P., Seder, R.A., Paul, W.E., Schwartz,
R.H., 1992. Activation events during thymic selection. J.
Exp. Med. 175, 731742.
De Martino, M., Rossi, M.E., Azzari, C., Gelli, M.G., Chiarelli, F.,
Galli, L., Vierucci, A., 1999. Viral load and CD69 molecule
expression on freshly isolated and cultured mitogen-stimulated
lymphocytes of children with perinatal HIV-1 infection. Clin.
Exp. Immunol. 117, 513516.
Green, S., Pichyangkul, S., Vaughn, D.W., Kalayanarooj, S.,
Nimmannitya, S., Nisalak, A., Kurane, I., Rothman, A.L.,
Ennis, F.A., 1999. Early CD69 expression on peripheral blood
lymphocytes from children with dengue hemorrhagic fever. J.
Infect. Dis. 180, 14291435.
Gubler, U., Hoffman, B.J., 1983. A simple and very efcient
method for generating cDNA libraries. Gene 25, 263269.
Hamann, J., Fiebig, H., Strauss, M., 1993. Expression cloning
of the early activation antigen CD69: a type II integral
membrane protein with a C-type lectin domain. J. Immunol.
150, 49204927.
Hara, T., Jung, L.K., Bjorndahl, J.M., Fu, S.M., 1986. Human
T cell activation. Part III. Rapid induction of a phosphorylated
J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 4348 47
28 kD/32 kD disulde-linked early activation antigen (EA 1) by
12-o-tetradecanoyl phorbol-13-acetate, mitogens, and antigens.
J. Exp. Med. 164, 19882005.
Hart, G.W., Brew, K., Grant, G.A., Bradshaw, R.A., Lennarz, W.J.,
1979. Primary structural requirements for the enzymatic
formation of the N-glycosidic bond in glycoproteins: studies
with natural and synthetic peptides. J. Biol. Chem. 254, 9747
9753.
Hartnell, A., Robinson, D.S., Kay, A.B., Wardlaw, A.J., 1993.
CD69 is expressed by human eosinophils activated in vivo in
asthma and in vitro by cytokines. Immunology 80, 281286.
Jung, L.K., Haynes, B.F., Nakamura, S., Pahwa, S., Fu, S.M., 1990.
Expression of early activation antigen (CD69) during human
thymic development. Clin. Exp. Immunol. 81, 466474.
Lanier, L.L., Buck, D.W., Rhodes, L., Ding, A., Evans, E., Barney,
C., Phillips, J.H., 1988. Interleukin 2 activation of natural
killer cells rapidly induces the expression and phosphorylation
oftheLeu-23activationantigen.J.Exp.Med.167,1572
1585.
Llera, A.S., Viedma, F., Sanchez-Madrid, F., Tormo, J., 2001.
Crystal structure of the C-type lectin-like domain from the
human hematopoietic cell receptor CD69. J. Biol. Chem. 276,
73127319.
Lopez-Cabrera, M., Santis, A.G., Fernandez-Ruiz, E., Blacher, R.,
Esch, F., Sanchez-Mateos, P., Sanchez-Madrid, F., 1993.
Molecular cloning, expression, and chromosomal localization
of the human earliest lymphocyte activation antigen AIM/
CD69: a new member of the C-type animal lectin superfamily
of signal-transmitting receptors. J. Exp. Med. 178, 537547.
Marzio, R., Mauel, J., Betz-Corradin, S., 1999. CD69 and
regulation of the immune function. Immunopharmacol. Im-
munotoxicol. 21, 565582.
Natarajan, K., Sawicki, M.W., Margulies, D.H., Mariuzza, R.A.,
2000. Crystal structure of human CD69: a C-type lectin-like
activation marker of hematopoietic cells. Biochemistry 39,
1477914786.
Nishikawa, K., Morii, T., Ako, H., Hamada, K., Saito, S., Narita,
N., 1992. In vivo expression of CD69 on lung eosinophils in
eosinophilic pneumonia: CD69 as a possible activation marker
for eosinophils. J. Allergy Clin. Immunol. 90, 169174.
Pinna, L.A., 1990. Casein kinase 2: an eminence grise in cellular
regulation? Biochim. Biophys. Acta. 1054, 267284.
Risso, A., Cosulich, M.E., Rubartelli, A., Mazza, M.R., Bargellesi,
A., 1989. MLR3 molecule is an activation antigen shared by
human B, T-lymphocytes and T cell precursors. Eur. J.
Immunol. 19, 323328.
Santis, A.G., Lopez-Cabrera, M., Sanchez-Madrid, F., Proudfoot,
N., 1995. Expression of the early lymphocyte activation antigen
CD69, a C-type lectin, is regulated by mRNA degradation
associated with AU-rich sequence motifs. Eur. J. Immunol. 25,
21422146.
Swat, W., Dessing, M., von Boehmer, H., Kisielow, P., 1993. CD69
expression during selection and maturation of CD4þ8þ
thymocytes. Eur. J. Immunol. 23, 739746.
Testi, R., Phillips, J.H., Lanier, L.L., 1989. Leu 23 induction as an
early marker of functional CD3/T cell antigen receptor
triggering. Requirement for receptor cross-linking, prolonged
elevation of intracellular [Ca
þþ
] and stimulation of protein
kinase C. J. Immunol. 142, 18541860.
Ullman, K.S., Northrop, J.P., Verweij, C.L., Crabtree, G.R., 1990.
Transmission of signals from the T-lymphocyte antigen
receptor to the genes responsible for cell proliferation and
immune function: the missing link. Ann. Rev. Immunol. 8,
421452.
Vance, B.A., Wu, W., Ribaudo, R.K., Segal, D.M., Kearse, K.P.,
1997. Multiple dimeric forms of human CD69 result from
differential addition of N-glycans to typical (AsnXSer/Thr)
and atypical (AsnXCys) glycosylation motifs. J. Biol. Chem.
272, 2311723122.
Woodgett, J.R., Gould, K.L., Hunter, T., 1986. Substrate specicity
of protein kinase C: use of synthetic peptides corresponding to
physiological sites as probes for substrate recognition require-
ments. Eur. J. Biochem. 161, 177184.
Ziegler, S.F., Ramsdell, F., Hjerrild, K.A., Armitage, R.J.,
Grabstein, K.H., Hennen, K.B., Farrah, T., Fanslow, W.C.,
Shevach, E.M., Alderson, M.R., 1993. Molecular characteriza-
tion of the early activation antigen CD69: a type II membrane
glycoprotein related to a family of natural killer cell activation
antigens. Eur. J. Immunol. 23, 16431648.
Ziegler, S.F., Levin, S.D., Johnson, L., Copeland, N.G., Gilbert,
D.J., Jenkins, N.A., Baker, E., Sutherland, G.R., Feldhaus, A.L.,
Ramsdell, F., 1994. The mouse CD69 gene: structure,
expression, and mapping to the NK gene complex. J. Immunol.
152, 12281236.
48 J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 4348
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