Cloning of bovine CD69
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
, M.J. Hamilton
, W.C. Davis
, Y.H. Park
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine,
Washington State University, Pullman, WA 99164-7040, USA
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
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
untranslated region, followed by a 600 bp 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. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: CD69; Cattle; Cloning; RACE; cDNA library
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
-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
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;
E-mail address: email@example.com (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 was collected from a Bos Taurus Holstein
by venipuncture of the jugular vein into 1/5 volume
acid–citrate–dextrose (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 3–6h
in RPMI supplemented with 2 mM
13% bovine serum.
2.2. cDNA library construction
Total RNA and mRNA were isolated using Trizol
(GIBCO BRL, Rockville, MD) and FastTrack
mRNA isolation kit (Invitrogen, Carlsbad, CA),
respectively. A cDNA library was constructed accord-
ing to the manufacturer’s protocol (Stratagene, La
Jolla, CA). In brief, 5 mg of mRNA was used to
synthesize the ﬁrst strand, and the second strand
was synthesized according to Gubler and Hoffman’s
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 puriﬁed cDNA was ligated with 1 mg of ZAP
Express vector (Stratagene). The titer of the primary
cDNA library was 1 10
pfu. Primary cDNA library
was ampliﬁed (6:0 10
pfu/ml) and used in PCR to
clone bovine CD69.
2.3. PCR ampliﬁcation of bovine CD69 through
Attempts to amplify bovine CD69 failed using two
degenerative primers. RACE (rapid ampliﬁcation of
cDNA ends) was applied to amplify bovine CD69 with
a highly consensus gene-speciﬁc primer and a ZAP
Express vector-speciﬁc primer. A gene-speciﬁc PCR
primer, BoCD69F 5
-GTG GGC CAATAC AAT TGT
was designed based on the conserved
amino acid sequence (VGQYNCPG) of human and
mouse CD69. It was used to amplify 3
-end of bovine
CD69 with a ZAP Express vector-speciﬁc primer,
-AAC GAC GGC CAG TGA ATT GT-3
The PCR reactions were performed in 100 ml volumes
containing 50 mM KCl, 10 mM Tris–HCl, pH 8.8,
1.5 mM MgCl
, 100 mM of each dNTP, 15 pmol of
each primer, 5 ml of ampliﬁed 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 ampliﬁcation of the
-end of bovine CD69; lane 3, PCR ampliﬁcation of the 5
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
potential rapid degradation signals are underlined.
J.S. Ahn et al. / Veterinary Immunology and Immunopathology 88 (2002) 43–48 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-speciﬁc primer, BoCD69R
-GCA TTT GCC CAC AGT TGT CAT A-3
designed downstream of stop codon and was used to
-end of bovine CD69 with a ZAP Express
vector-speciﬁc primer, ZAPF 5
-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) 43–48
CGC CAA GC-3
. 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
3–6 h. Because ampliﬁcation of bovine CD69 failed
using two degenerative primers, we constructed a
cDNA library. RACE was successful in amplifying
-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
-end of bovine CD69,
a gene-speciﬁc primer was designed and used to
-end of bovine CD69 with another ZAP
Express-speciﬁc 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
-end untranslated region, an open reading frame
(ORF) of 199 amino acids and potential AU-rich
motifs in the 3
-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 unidentiﬁed
ligand for bovine CD69. Human and mouse CD69
have three intra-chain disulﬁde bonds, Cys
, and Cys
(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
(Hamann et al.,
1993). Because Cys
precedes ﬁrst b0 strand and
is located within b1 strand at the N-terminus,
does not change the structure of bovine CD69
signiﬁcantly (Natarajan et al., 2000; Llera et al.,
2001). Bovine CD69 contains two typical glycosyla-
tion sites (N–X–S/T; Hart et al., 1979) and one aty-
pical glycosylation site (N–X–C) 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/T–X–X–D/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/T–X–K/R; Woodgett et al.,
1986). These observations suggest that bovine CD69
may be important for the activation of lymphocytes.
This work was supported in part by Grants from the
USDA-NRICGP no. 98-02-02480, WNV-00138 and
Brain Korea 21 project.
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