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Cloning and structural analysis of equine platelet endothelial cell
adhesion molecule (PECAM, CD31) and vascular cell
adhesion molecule-1 (VCAM-1, CD106)
Abigail J. Gregg, Alan R. Schenkel*
Department of Microbiology, Immunology & Pathology, College of Veterinary and Biomedical Sciences, Colorado State University,
1682 Campus Delivery Fort Collins, CO 80523-1682, United States
Received 11 October 2007; received in revised form 13 November 2007; accepted 15 November 2007
Platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106) are
essential for leukocyte emigration and diapedesis. PECAM is an essential histologic marker of endothelial cells; VCAM-1 is a
prototype marker forendothelial cell activation. In this study, equine PECAM and VCAM mRNAwere cloned and sequenced. Both
genes are highly conserved amongst several species. This study also revealed conserved structural and regulatory motifs,
emphasizing the importance of these genes’ physiological roles in immunological responses.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Equine; Endothelial cell; Leukocyte; Adhesion molecule; Extravasation; Diapedesis
(PECAM, CD31) and vascular cell adhesion mole-
cule-1 (VCAM-1, CD106) are essential for leukocyte
emigration and diapedesis. PECAM is constitutively
expressed by endothelial cells, platelets, and most
leukocytes, whereas VCAM is induced on endothelial
cells at sites of inflammation. PECAM on endothelial
cells binds to PECAM on leukocytes to facilitate
diapedesis. VCAM-1 is the ligand for the leukocyte
CD49d/CD29 integrin, also known as very late antigen-
Damle and Aruffo, 1991; Muller, 1995, 1999; Newman
et al., 1990; Oppenheimer-Marks et al., 1991; Osborn
et al., 1989; Schenkel et al., 2004).
Equine PECAM and VCAM are essential identifica-
tion and inflammatory activation markers of endothelial
cells, yet have not previously been described in the
horse. We cloned and sequenced mRNA from horse
tracheo-bronchial lymph nodes. Primers were designed
based on the equine contigs that had human sequence
homology in the 50and 30UTRs of both PECAM and
VCAM (Table 1). Then the sequence was completed
from both ends using primers from confirmed sequence
designed with Primer3 software (http://primer3.source-
forge.net/). Full-length clones were isolated using the
Topo TAcloningsystem (Invitrogen, Carlsbad, CA) and
sequenced overlapping several regions. Sequences were
numbers: PECAM EU031642 and VCAM EU057895).
PECAM hastwoin-frame AUGstartcodons (Fig.1),
but equine VCAM does not (Fig. 2). In rodents the first
in-frame ‘‘start’’ codon of PECAM is not present but in
horse, human, cattle, and dog the sequence is retained
Available online at www.sciencedirect.com
Veterinary Immunology and Immunopathology 122 (2008) 295–308
* Corresponding author. Tel.: +1 970 491 2926;
fax: +1 970 491 1815.
E-mail address: email@example.com (A.R. Schenkel).
0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
Author's personal copy
A.J. Gregg, A.R. Schenkel/Veterinary Immunology and Immunopathology 122 (2008) 295–308296
Fig. 1. PECAM mRNA and amino acid sequence. Signal peptide is in italics. Immunoglobulin domains 1–6 are each enclosed by brackets. The
transmembrane portion is indicated by underline. Signal tyrosines are indicated by asterisks.
Author's personal copy
The future goals of this project are to produce
proteins for immunization and subsequent polyclonal
and monoclonal antibody production. These reagents
will be extremely useful for not just immunological
studies, but also in histology as these markers are
amongst the best characterized in the scientific
literature.These markers are alsouseful in angiogenesis
A.J. Gregg, A.R. Schenkel/Veterinary Immunology and Immunopathology 122 (2008) 295–308307
Fig. 6. Unrooted phylogenetic map of VCAM between horse, human, dog, cow, rat and mouse. See legend of Fig. 5 for more information.
Fig. 5. Unrootedphylogeneticmap of PECAMbetweenhorse, human, dog,cow, rat and mouse. Relativemap distancesare indicated by numbersat
each position. All analysis done using the online tools at the European Bioinformatics Institute of the European Molecular Biology Laboratories
(http://www.ebi.ac.uk/) (Kimura, 1983; Nei, 1972).
Fig. 7. Conserved regulatory elements of the VCAM promoter analyzed from the contig AAWR01013283 and compared to the human promoter
region (Iademarco et al., 1992). All transcription factors are in bold and underlined. The putative transcription start site is indicated by +1.
Fig. 4. (Continued).
Author's personal copy
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Homology across several species
Genbank accession numbers for nucleotide comparisons: PECAM
NM_008816/NM_001032378, Dog XM_848326.1. VCAM: Human
NM_001078.1, Bovine NM_174484.1, Rat NM_012889.1, Mouse
NM_011693.2, Dog NM_0010023298.1.