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CD11b of Ovis canadensis and Ovis aries: Molecular cloning and characterization
Veterinary Immunology and Immunopathology 119 (2007) 287–298 www.elsevier.com/locate/vetimm
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CD11b of Ovis canadensis and Ovis aries: Molecular cloning and characterization Paulraj K. Lawrence, Subramaniam Srikumaran * Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA Received 19 April 2007; accepted 24 May 2007
Abstract Leukotoxin (Lkt) is the primary virulence factor secreted by Mannheimia haemolytica which causes pneumonia in ruminants. Previously, we have shown that CD18, the b subunit of b2 integrins, mediates Lkt-induced cytolysis of ruminant leukocytes. CD18 associates with four distinct a subunits giving rise to four b2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/ CD18 (CR4), and CD11d/CD18. It is not known whether all the b2 integrins serve as a receptor for Lkt. Since PMNs are the leukocyte subset that is most susceptible to Lkt, and Mac-1 expression on PMNs exceeds that of other b2 integrins, it is of interest to determine whether Mac-1 serves as a receptor for Lkt which necessitates the cloning of CD11b and CD18. In this study, we cloned and sequenced the cDNA encoding CD11b of Ovis canadensis (bighorn sheep) and Ovis aries (domestic sheep). CD11b cDNA is 3455 nucleotides long encoding a polypeptide of 1152 amino acids. CD11b polypeptides from these two species exhibit 99% identity with each other, and 92% with that of cattle, and 70–80% with that of the non-ruminants analyzed. # 2007 Elsevier B.V. All rights reserved. Keywords: Bighorn sheep; Domestic sheep; CD11b; cDNA; Cloning
Integrins are adhesion molecules found on most cell types and facilitate intercellular communications. These are heterodimeric molecules composed of a and b chains (Pribila et al., 2004). The integrins have been classified into subfamilies depending upon which b subunit they contain (Gahmberg et al., 1998). b2 subfamily of integrins are unique to leukocytes. The b2 integrins have a common b subunit, CD18 that associates with three distinct a chains, CD11a, CD11b, and CD11c to give rise to three different b2 integrins: CD11a/CD18, also known as LFA-1 (lymphocyte function-associated antigen 1); CD11b/CD18, also known as Mac-1 and CR3 (complement receptor 3); CD11c/CD18 also known as CR4 and p150/95. The
* Corresponding author. Tel.: +1 509 335 4572; fax: +1 509 335 8529. E-mail address: [email protected] (S. Srikumaran). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.05.019
more recently identified b2 integrin CD11d/CD18 (Noti et al., 2000) is not well characterized in ruminants yet. The relative amounts of CD11/CD18 antigens expressed, depends on the cell type and the state of differentiation and activation. LFA-1 is expressed on all leukocytes. Mac-1 is enriched on PMNs, but is also expressed on monocytes, macrophages, NK cells and certain subpopulations of B lymphocytes. CR4 is enriched on monocytes and macrophages, but is also expressed on PMNs and a small subpopulation of B cells (Gahmberg et al., 1998). Both a and b subunits are required for the expression of the heterodimer on the cell surface. These molecules facilitate homing into areas of inflammation, phagocytosis, antigen presentation and cytotoxicity, by interacting with various intercellular adhesion molecules (ICAMs) expressed by the leukocytes, endothelial cells, and many other tissues, and with soluble proteins such as the complement fragments and fibrinogen (Gahmberg et al., 1998).
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Activated Mac-1 efficiently binds to wide array of ligands including numerous, intercellular adhesion molecules (ICAM-1 and ICAM-2), extracellular matrix proteins and blood coagulation proteins. Several pathogens including Mycobacterium tuberculosis (Cywes et al., 1996), Neisseria gonorrhaeace (Edwards and Apicella, 2005), Bordetella pertussis (Relman et al., 1990), Candida albicans (Forsyth and Mathews, 1996), Leishmania species (Rosenthal et al., 1996) and Blastomyces dermatitidis (Newman et al., 1995) utilize Mac-1 as their receptor on host cells. Earlier studies in our laboratory have revealed that b2 integrins serve as the receptor for leukotoxin (Lkt) of Mannheimia haemolytica which causes pneumonia in bighorn sheep (BHS, Liu et al., 2007) and domestic sheep (DS, Dassanayake et al., 2007). Lkt secreted by M. haemolytica is an important virulence factor of this organism, and is cytotoxic to all the leukocyte subsets of BHS and DS. Although our studies clearly indicated that CD18, the b subunit of b2 integrins, mediates Lktinduced cytolysis of target cells, it is not clear whether all the b2 integrins serve as a receptor for Lkt. In recent studies, we have demonstrated that LFA-1 of BHS and DS serves as a receptor for Lkt (Dassanayake et al., submitted for publication; Lawrence et al., 2007). Since PMNs are the leukocyte subset that is most susceptible to Lkt-induced cytolysis, and Mac-1 expression on PMNs exceeds that of other b2 integrins (Arnaout, 1990), Mac-1 is likely to serve as a receptor for Lkt. Clarification of the role of Mac-1 in Lkt-induced cytolysis, in the absence of any compounding effects of the other b2 integrins, necessitates the co-expression of CD11b and CD18 in a cell-line that does not express any b2 integrins. This approach requires the cloning and sequencing of cDNA encoding CD11b and CD18. We have previously cloned and sequenced the cDNA encoding CD18 of BHS (Liu et al., 2006) and DS (Dassanayake et al., 2007). The next logical step would be to clone the cDNA encoding CD11b of BHS and DS. PMNs were isolated from peripheral blood by density gradient centrifugation as described earlier (Deshpande et al., 2002). The total RNA from PMNs was extracted using TRIzol reagent and cDNA was synthesized using Superscript IIITM first-strand synthesis kit following the manufacturer’s instructions (Invitrogen Inc., Carlsbad, CA). Forward and reverse primers to amplify the CD11b gene was designed based on multiple alignment of human (NM 000632), rat (AF 268593) and mouse (NM 008401) cDNA sequences available in the GenBank. The primers designed were: CD11b For; 50 -GTTCTGGCTCCTTCCAGCCATGGCTCTCAGAGTCCTTCTG-30 and CD11b Rev; 50 -
GAGAGGCAGCTCTGTCGGGAAGGAGCCGCTACTGGGGT-30 . Since, we could not amplify the complete CD11b gene as a single fragment by using the above two primers, we designed two additional primers from the internal N-terminal consensus region. CD11b F1 For; 50 -CCTCTACCACTGTGACTACAGCAC-30 and CD11b F1 Rev; 50 -GTGCTGTAGTCACAGTGG TAGAGG-30 . By using the combination of CD11b F1 and CD11b Rev 3274 nt of CD11b gene was amplified and by using CD11b For and CD11b F1 Rev the remaining 210 nt N-terminal fragment was amplified. The CD11b gene fragments were amplified using a high fidelity polymerase, PfuUltraTM II Fusion HS (Stratagene, La Jolla, CA). The PCR fragments were gelpurified and cloned into pCR14 Blunt-TOPO vector (Invitrogen). The positive clones containing insert were identified by colony PCR and sequenced completely. Thus, the cloned CD11b gene was in two fragments. These two fragments had 26 nucleotide overlap between them, but no unique restriction enzyme site that could be used to join these fragments. Restriction analysis revealed a unique internal PstI at 232 bp position from the start codon, but in a non-overlapping region between these fragments. In order to avoid the addition of any restriction sites to join these fragments, which would result in the addition of at least two amino acids we synthesized the 246 bp N-terminal region of the gene (Genemed Synthesis, Inc., San Francisco, CA) by adding a PstI site preceding the ATG including the naturally available PstI site. This fragment was cloned into pUC57 at PstI site. This was made possible because we had the complete gene sequences from both these species and they were 100% identical in this region. Since the pCR4 Blunt vector has an unique PstI site, in addition to the PstI site already available on the 3274 bp CD11b gene fragment, digestion with PstI would release the fragment, making it impossible to join it with the 246 bp N-terminal fragment. We wanted to create a recombinant construct with only one naturally available PstI site within the gene and eliminate any site in the vector. In order to achieve this objective, the 3274 bp fragment was released from the pCR4 Blunt vector back bone after double digestion with EcoRI and PstI, gel-purified and ligated into pUC19 using clonables mix (EMD Biosciences, Inc., San Diego, CA) and transformed into TOP10 cells (Invitrogen). The positive clones were sequenced to confirm the junction between insert and vector. The pUC19 construct harboring the 3274 bp fragment was digested with PstI, dephosphorylated using shrimp alkaline
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phosphatase (Fermentas Inc., Hanover, MD) and the 246 bp N-terminal fragment, after digestion with PstI was ligated to 3274 bp by the procedure described above. The orientation of the insert (246 bp) in the final
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construct was confirmed by sequencing. Now we had the full length CD11b gene cloned into pUC19 vector. Figs. 1 and 2 show the complete nucleotide and derived amino acid sequence of BHS and DS CD11b (GenBank
Fig. 1. Nucleotide and deduced amino acid sequence of BHS CD11b. The predicted features are indicated by the symbols: N-terminal signal sequence (solid line); transmembrane domain (@); SEC7 region (); putative N-glycosylation sites (*).
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accession numbers EF206309 and EF206308, respectively). Sequencing reactions were performed by Big DyeTM (Applied Biosystems, CA) and analyzed by BioEdit v7.0.5 (Ibis therapeutics, Carlsbad, CA). BLASTn and
BLAST 2 SEQUENCES (http://www.ncbi.nlm.nih.gov/) were utilized for homology and identity %; open reading frames were confirmed by ExPASy (http:// us.expasy.org/) and CLUSTAL_W 1.8 (http://www.ebi.ac.uk) was employed align the amino acid sequences
Fig. 1. (Continued ).
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Fig. 1. (Continued ).
(Thompson et al., 1994). Protein statistics were analyzed by EMBOSS server (http://www.bioinformatics2.wsu.edu/emboss/). The isolated BHS and DS CD11b genes were identical in length, i.e., 3455 bp long encoding 1152 aa (analyzed three clones from each species). The deduced polypeptide was 99% identical with respect to each other. The substitution of aa in DS CD11b and their positions with respect to BHS are as follows: L211P, N243S, H381Y, G487R, P513L, A526V, T599S, R699L, M1001T and V1024I. Out of these 10 substitutions, the first 2 falls in the insertion (I)/von Willebrand factor A domain, which is involved in adhesive interactions (Lee et al., 1995). The substitution
A526V, seems to be identical across species with alanine at this position in BHS and cattle, and valine in DS, humans, rats, and mice. BHS and DS CD11b have an N-terminal signal peptide sequence between 16 and 17 aa (maximum probability of 0.963). The predicted transmembrane domain is 23 aa long, which is followed by an integrin alpha chain signature sequence, GFFKR which is 100% identical among the species analyzed. The comparison of CD11b of BHS and DS with that of other species is shown in Table 1. Even though the maximum % identity at nt level is with rats, CD11b polypeptide shares lowest aa % identity with rats and mice but maximum with cattle. The overall protein structure and domains are in agreement with previously
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isolated bovine CD11b (Gopinath et al., 2006). As seen in Table 2, the predicted CD11b polypeptide of BHS and DS are identical with respect to most characteristics. They have similar molar composition of charged, basic, acidic, polar and non-polar amino acids, which
suggests possible functional similarities with respect to ligand binding. The I-domain found in various plasma protein complement factors (Hogg et al., 1994; Emsley et al., 1998) spans between 150 and 350 aa and is similar to human, cattle, rat and mouse. The domain adopts a
Fig. 2. Nucleotide and deduced amino acid sequence of DS CD11b. The predicted features are indicated by the symbols: N-terminal signal sequence (solid line); transmembrane domain (@); SEC7 region (); putative N-glycosylation sites (*).
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Fig. 2. (Continued ).
classic a/b Rossmann fold and contains a metal iondependent adhesion site (MIDAS) for binding protein ligands (Lee et al., 1995; Qu and Leahy, 1995). The residues (DXSXS) constituting the MIDAS motif in BHS and DS CD11b are completely conserved
and is 100% identical across species compared (Fig. 3). Both BHS and DS contain, 14 predicted N-linked glycosylation sites (Asn-X-Ser/Thr) at identical locations similar to cattle CD11b (Gopinath et al., 2006) as
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Fig. 2. (Continued ).
Table 1 Comparison of CD11b of BHS and DS with that of other species Species/GenBank#
% Identity Bighorn sheep
Cattle (NM 001039957.1) Humans (BC 099660) Chimpanzees (XM 510949) Pigs (U40072) Dogs (XM 843434) Rats (NM 012711) Mice (NM 008401)
Domestic sheep
Nucleotide
Amino acid
Nucleotide
Amino acid
95 88 88 86 87 96 92
92 74 74 80 73 70 70
95 88 88 86 87 96 92
92 74 75 80 73 71 71
The table shows % identity at nucleotide and predicted amino acid levels.
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Fig. 3. Comparison of predicted amino acid sequence of BHS and DS CD11b with that of humans (HuCD11b, BC 099660), cattle (BoCD11b, NM 001039957.1), mice (MoCD11b, NM008401) and rats (RaCD11b, NM012711). The predicted features are indicated by the symbols: I-domain (*); MIDAS motif ($); integrin alpha signature sequence (+); FG-GAP repeats (=); divalent cation binding motif (^).
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Fig. 3. (Continued ).
P.K. Lawrence, S. Srikumaran / Veterinary Immunology and Immunopathology 119 (2007) 287–298 Table 2 Predicted protein characteristics of BHS and DS CD11b
No. of aa Predicted molecular mass (Da) Isoelectric point (pI) Overall charge N-Glycosylation sites Divalent cation binding sites
Bighorn sheep
Domestic sheep
1152 127550.87 7.57 13.5 14 3
1152 127603.95 7.52 13.5 14 3
predicted by NetNGlyc 1.0 server (http:// www.cbs.dtu.dk/services/NetNGlyc/). The BHS and DS CD11b contain seven FG-GAP repeats as seen in cattle, which are predicted to be b propeller structures. There are three potential divalent cation (Ca2+, Mg2+ and Mn2+) binding sites (DX[DN]XDXXXD) which are highly conserved among species and is identical to humans, rats, mice and cattle. BHS and DS CD11b also contain 22 cysteine residues at identical positions, possibly involved in the formation of disulfide bridges. The aa from 63 to 218 are homologous to Saccharomyces cerevisiae SEC7 gene product (Achstetter et al., 1988), which is required for proper protein transport through the Golgi apparatus for post-translational modification. The aa between 343 and 382 contain WD-40 repeats (Neer et al., 1994) which form a circularized beta-propeller structure, necessary for coordinating multi-protein complex assemblies which resonates with the functional properties of integrins. The potential S, T and Y phosphorylation sites are spread all over the CD11b polypeptide, but the cytoplasmic tail contains equal number of S/T phosphorylation sites located at identical positions in both these species. The phylogenetic tree as expected shows a close relationship between BHS and DS CD11b compared to other species (Fig. 4).
Fig. 4. Phylogenetic analysis of CD11b of BHS, DS, and other species. Phylogenetic tree was constructed by neighbor-joining using default settings by online program http://align.genome.jp/sit-bin/clustalw.
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Availability of cDNA for CD11b and CD18 from BHS and DS will enable the development of transfectants expressing recombinant Mac-1 and clarify its role in M. haemolytica-induced cyotoxicity. Acknowledgments This research was supported by funds from the Foundation for North American Wild Sheep and its Eastern, Idaho, Oregon, and Washington Chapters. References Achstetter, T., Franzusoff, A., Field, C., Schekman, R., 1988. SEC7 encodes an unusual, high molecular weight protein required for membrane traffic from the yeast Golgi apparatus. J. Biol. Chem. 263, 11711–11717. Arnaout, M.A., 1990. Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75, 1037–1050. Cywes, C., Godenir, N.L., Hoppe, H.C., Scholle, R.R., Steyn, L.M., Kirsch, R.E., Ehlers, M.R., 1996. Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells. Infect. Immun. 64, 5373–5383. Dassanayake, R.P., Shantalingam, S., Davis, W.C., Srikumaran, S., 2007. Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the b subunit of b2 integrins. Microb. Pathog. (Epub ahead of print). Dassanayake, R.P., Liu, W., Davis, W.C., Foreyt, W.J., Srikumaran, S., submitted for publication. Recombinant expression of Ovis canadensis lymphocyte function-associated antigen-1 in non-susceptible cells identifies it as a receptor for Mannheimia haemolytica leukotoxin. Deshpande, M.S., Ambagala, T.C., Ambagala, A.P., Kehrli Jr., M.E., Srikumaran, S., 2002. Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxininduced cytolysis. Infect. Immun. 70, 5058–5064. Edwards, J.L., Apicella, M.A., 2005. I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells. Cell. Microbiol. 7, 1197–1211. Emsley, J., Cruz, M., Handin, R., Liddington, R., 1998. Crystal structure of the von Willebrand Factor A1 domain and implications for the binding of platelet glycoprotein Ib. J. Biol. Chem. 273, 10396–10401. Forsyth, C.B., Mathews, H.L., 1996. Lymphocytes utilize CD11b/ CD18 for adhesion to Candida albicans. Cell. Immunol. 170, 91–100. Gahmberg, C.G., Valmu, L., Fagerholm, S., Kotovuori, P., Ihanus, E., Tian, L., Pessa-Morokawa, T., 1998. Leukocyte integrins and inflammation. Cell. Mol. Life Sci. 54, 549–555. Gopinath, R.S., Ambagala, A.P.N., Ambagala, T.C., Liu, W., Srikumaran, S., 2006. Molecular cloning and characterization of cDNA encoding CD11b of cattle. Vet. Immunol. Immunopathol. 110, 349–355. Hogg, N., Landis, C.R., Bates, P.A., Stanley, P., Randi, A.M., 1994. The sticking point: how integrins bind to their ligands. Trends Cell Biol. 4, 379–382. Lawrence, P.K., Dassanayake, R.P., Knowles, D.P., Srikumaran, S., 2007. Transfection of non-susceptible cells with Ovis aries
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recombinant lymphocyte function-associated antigen 1 renders susceptibility to Mannheimia haemolytica leukotoxin. Vet. micro., doi:10.1016/j.vetmic.2007.05.006. Lee, J.O., Rieu, P., Arnaout, M.A., Liddington, R., 1995. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80, 631–638. Liu, W., Brayton, K.A., Lagerquist, J., Foreyt, W.J., Srikumaran, S., 2006. Cloning and comparison of bighorn sheep CD18 with that of domestic sheep, goats, cattle, humans and mice. Vet. Immunol. Immunopathol. 110, 11–16. Liu, W., Brayton, K.A., Davis, W.C., Mansfield, K., Lagerquist, J., Foreyt, W.J., Srikumaran, S., 2007. Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes. J. Wildl. Dis. 43, 75–81. Neer, E.J., Schmidt, C.J., Nambudripad, R., Smith, T.F., 1994. The ancient regulatory-protein family of WD-repeat proteins. Nature 371, 297–300. Newman, S.L., Chaturvedi, S., Klein, B.S., 1995. The WI-1 antigen of Blastomyces dermatitidis yeasts mediates binding to human macrophage CD11b/CD18 (CR3) and CD14. J. Immunol. 154, 753–761.
Noti, J.D., Johnson, A.K., Dillon, J.D., 2000. Structural and functional characterization of the leukocyte integrin gene CD11d. Essential role of sp1 and Sp3. J. Biol. Chem. 275, 8959–8969. Pribila, J.T., Quale, A.C., Mueller, K.L., Shimizu, Y., 2004. Integrins and T cell mediated immunity. Annu. Rev. Immunol. 22, 157–180. Qu, A., Leahy, D.J., 1995. Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, alpha L beta 2) integrin. Proc. Natl. Acad. Sci. U.S.A. 92, 10277–10281. Relman, D., Tuomanen, E., Falkow, S., Golenbock, D.T., Saukkonen, K., Wright, S.D., 1990. Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 61, 1375– 1382. Rosenthal, L.A., Sutterwala, F.S., Kehrli, M.E., Mosser, D.M., 1996. Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion. Infect. Immun. 64, 2206–2215. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
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CD11b of Ovis canadensis and Ovis aries: Molecular cloning and characterization Paulraj K. Lawrence, Subramaniam Srikumaran * Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA Received 19 April 2007; accepted 24 May 2007
Abstract Leukotoxin (Lkt) is the primary virulence factor secreted by Mannheimia haemolytica which causes pneumonia in ruminants. Previously, we have shown that CD18, the b subunit of b2 integrins, mediates Lkt-induced cytolysis of ruminant leukocytes. CD18 associates with four distinct a subunits giving rise to four b2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/ CD18 (CR4), and CD11d/CD18. It is not known whether all the b2 integrins serve as a receptor for Lkt. Since PMNs are the leukocyte subset that is most susceptible to Lkt, and Mac-1 expression on PMNs exceeds that of other b2 integrins, it is of interest to determine whether Mac-1 serves as a receptor for Lkt which necessitates the cloning of CD11b and CD18. In this study, we cloned and sequenced the cDNA encoding CD11b of Ovis canadensis (bighorn sheep) and Ovis aries (domestic sheep). CD11b cDNA is 3455 nucleotides long encoding a polypeptide of 1152 amino acids. CD11b polypeptides from these two species exhibit 99% identity with each other, and 92% with that of cattle, and 70–80% with that of the non-ruminants analyzed. # 2007 Elsevier B.V. All rights reserved. Keywords: Bighorn sheep; Domestic sheep; CD11b; cDNA; Cloning
Integrins are adhesion molecules found on most cell types and facilitate intercellular communications. These are heterodimeric molecules composed of a and b chains (Pribila et al., 2004). The integrins have been classified into subfamilies depending upon which b subunit they contain (Gahmberg et al., 1998). b2 subfamily of integrins are unique to leukocytes. The b2 integrins have a common b subunit, CD18 that associates with three distinct a chains, CD11a, CD11b, and CD11c to give rise to three different b2 integrins: CD11a/CD18, also known as LFA-1 (lymphocyte function-associated antigen 1); CD11b/CD18, also known as Mac-1 and CR3 (complement receptor 3); CD11c/CD18 also known as CR4 and p150/95. The
* Corresponding author. Tel.: +1 509 335 4572; fax: +1 509 335 8529. E-mail address: [email protected] (S. Srikumaran). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.05.019
more recently identified b2 integrin CD11d/CD18 (Noti et al., 2000) is not well characterized in ruminants yet. The relative amounts of CD11/CD18 antigens expressed, depends on the cell type and the state of differentiation and activation. LFA-1 is expressed on all leukocytes. Mac-1 is enriched on PMNs, but is also expressed on monocytes, macrophages, NK cells and certain subpopulations of B lymphocytes. CR4 is enriched on monocytes and macrophages, but is also expressed on PMNs and a small subpopulation of B cells (Gahmberg et al., 1998). Both a and b subunits are required for the expression of the heterodimer on the cell surface. These molecules facilitate homing into areas of inflammation, phagocytosis, antigen presentation and cytotoxicity, by interacting with various intercellular adhesion molecules (ICAMs) expressed by the leukocytes, endothelial cells, and many other tissues, and with soluble proteins such as the complement fragments and fibrinogen (Gahmberg et al., 1998).
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Activated Mac-1 efficiently binds to wide array of ligands including numerous, intercellular adhesion molecules (ICAM-1 and ICAM-2), extracellular matrix proteins and blood coagulation proteins. Several pathogens including Mycobacterium tuberculosis (Cywes et al., 1996), Neisseria gonorrhaeace (Edwards and Apicella, 2005), Bordetella pertussis (Relman et al., 1990), Candida albicans (Forsyth and Mathews, 1996), Leishmania species (Rosenthal et al., 1996) and Blastomyces dermatitidis (Newman et al., 1995) utilize Mac-1 as their receptor on host cells. Earlier studies in our laboratory have revealed that b2 integrins serve as the receptor for leukotoxin (Lkt) of Mannheimia haemolytica which causes pneumonia in bighorn sheep (BHS, Liu et al., 2007) and domestic sheep (DS, Dassanayake et al., 2007). Lkt secreted by M. haemolytica is an important virulence factor of this organism, and is cytotoxic to all the leukocyte subsets of BHS and DS. Although our studies clearly indicated that CD18, the b subunit of b2 integrins, mediates Lktinduced cytolysis of target cells, it is not clear whether all the b2 integrins serve as a receptor for Lkt. In recent studies, we have demonstrated that LFA-1 of BHS and DS serves as a receptor for Lkt (Dassanayake et al., submitted for publication; Lawrence et al., 2007). Since PMNs are the leukocyte subset that is most susceptible to Lkt-induced cytolysis, and Mac-1 expression on PMNs exceeds that of other b2 integrins (Arnaout, 1990), Mac-1 is likely to serve as a receptor for Lkt. Clarification of the role of Mac-1 in Lkt-induced cytolysis, in the absence of any compounding effects of the other b2 integrins, necessitates the co-expression of CD11b and CD18 in a cell-line that does not express any b2 integrins. This approach requires the cloning and sequencing of cDNA encoding CD11b and CD18. We have previously cloned and sequenced the cDNA encoding CD18 of BHS (Liu et al., 2006) and DS (Dassanayake et al., 2007). The next logical step would be to clone the cDNA encoding CD11b of BHS and DS. PMNs were isolated from peripheral blood by density gradient centrifugation as described earlier (Deshpande et al., 2002). The total RNA from PMNs was extracted using TRIzol reagent and cDNA was synthesized using Superscript IIITM first-strand synthesis kit following the manufacturer’s instructions (Invitrogen Inc., Carlsbad, CA). Forward and reverse primers to amplify the CD11b gene was designed based on multiple alignment of human (NM 000632), rat (AF 268593) and mouse (NM 008401) cDNA sequences available in the GenBank. The primers designed were: CD11b For; 50 -GTTCTGGCTCCTTCCAGCCATGGCTCTCAGAGTCCTTCTG-30 and CD11b Rev; 50 -
GAGAGGCAGCTCTGTCGGGAAGGAGCCGCTACTGGGGT-30 . Since, we could not amplify the complete CD11b gene as a single fragment by using the above two primers, we designed two additional primers from the internal N-terminal consensus region. CD11b F1 For; 50 -CCTCTACCACTGTGACTACAGCAC-30 and CD11b F1 Rev; 50 -GTGCTGTAGTCACAGTGG TAGAGG-30 . By using the combination of CD11b F1 and CD11b Rev 3274 nt of CD11b gene was amplified and by using CD11b For and CD11b F1 Rev the remaining 210 nt N-terminal fragment was amplified. The CD11b gene fragments were amplified using a high fidelity polymerase, PfuUltraTM II Fusion HS (Stratagene, La Jolla, CA). The PCR fragments were gelpurified and cloned into pCR14 Blunt-TOPO vector (Invitrogen). The positive clones containing insert were identified by colony PCR and sequenced completely. Thus, the cloned CD11b gene was in two fragments. These two fragments had 26 nucleotide overlap between them, but no unique restriction enzyme site that could be used to join these fragments. Restriction analysis revealed a unique internal PstI at 232 bp position from the start codon, but in a non-overlapping region between these fragments. In order to avoid the addition of any restriction sites to join these fragments, which would result in the addition of at least two amino acids we synthesized the 246 bp N-terminal region of the gene (Genemed Synthesis, Inc., San Francisco, CA) by adding a PstI site preceding the ATG including the naturally available PstI site. This fragment was cloned into pUC57 at PstI site. This was made possible because we had the complete gene sequences from both these species and they were 100% identical in this region. Since the pCR4 Blunt vector has an unique PstI site, in addition to the PstI site already available on the 3274 bp CD11b gene fragment, digestion with PstI would release the fragment, making it impossible to join it with the 246 bp N-terminal fragment. We wanted to create a recombinant construct with only one naturally available PstI site within the gene and eliminate any site in the vector. In order to achieve this objective, the 3274 bp fragment was released from the pCR4 Blunt vector back bone after double digestion with EcoRI and PstI, gel-purified and ligated into pUC19 using clonables mix (EMD Biosciences, Inc., San Diego, CA) and transformed into TOP10 cells (Invitrogen). The positive clones were sequenced to confirm the junction between insert and vector. The pUC19 construct harboring the 3274 bp fragment was digested with PstI, dephosphorylated using shrimp alkaline
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phosphatase (Fermentas Inc., Hanover, MD) and the 246 bp N-terminal fragment, after digestion with PstI was ligated to 3274 bp by the procedure described above. The orientation of the insert (246 bp) in the final
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construct was confirmed by sequencing. Now we had the full length CD11b gene cloned into pUC19 vector. Figs. 1 and 2 show the complete nucleotide and derived amino acid sequence of BHS and DS CD11b (GenBank
Fig. 1. Nucleotide and deduced amino acid sequence of BHS CD11b. The predicted features are indicated by the symbols: N-terminal signal sequence (solid line); transmembrane domain (@); SEC7 region (); putative N-glycosylation sites (*).
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accession numbers EF206309 and EF206308, respectively). Sequencing reactions were performed by Big DyeTM (Applied Biosystems, CA) and analyzed by BioEdit v7.0.5 (Ibis therapeutics, Carlsbad, CA). BLASTn and
BLAST 2 SEQUENCES (http://www.ncbi.nlm.nih.gov/) were utilized for homology and identity %; open reading frames were confirmed by ExPASy (http:// us.expasy.org/) and CLUSTAL_W 1.8 (http://www.ebi.ac.uk) was employed align the amino acid sequences
Fig. 1. (Continued ).
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Fig. 1. (Continued ).
(Thompson et al., 1994). Protein statistics were analyzed by EMBOSS server (http://www.bioinformatics2.wsu.edu/emboss/). The isolated BHS and DS CD11b genes were identical in length, i.e., 3455 bp long encoding 1152 aa (analyzed three clones from each species). The deduced polypeptide was 99% identical with respect to each other. The substitution of aa in DS CD11b and their positions with respect to BHS are as follows: L211P, N243S, H381Y, G487R, P513L, A526V, T599S, R699L, M1001T and V1024I. Out of these 10 substitutions, the first 2 falls in the insertion (I)/von Willebrand factor A domain, which is involved in adhesive interactions (Lee et al., 1995). The substitution
A526V, seems to be identical across species with alanine at this position in BHS and cattle, and valine in DS, humans, rats, and mice. BHS and DS CD11b have an N-terminal signal peptide sequence between 16 and 17 aa (maximum probability of 0.963). The predicted transmembrane domain is 23 aa long, which is followed by an integrin alpha chain signature sequence, GFFKR which is 100% identical among the species analyzed. The comparison of CD11b of BHS and DS with that of other species is shown in Table 1. Even though the maximum % identity at nt level is with rats, CD11b polypeptide shares lowest aa % identity with rats and mice but maximum with cattle. The overall protein structure and domains are in agreement with previously
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isolated bovine CD11b (Gopinath et al., 2006). As seen in Table 2, the predicted CD11b polypeptide of BHS and DS are identical with respect to most characteristics. They have similar molar composition of charged, basic, acidic, polar and non-polar amino acids, which
suggests possible functional similarities with respect to ligand binding. The I-domain found in various plasma protein complement factors (Hogg et al., 1994; Emsley et al., 1998) spans between 150 and 350 aa and is similar to human, cattle, rat and mouse. The domain adopts a
Fig. 2. Nucleotide and deduced amino acid sequence of DS CD11b. The predicted features are indicated by the symbols: N-terminal signal sequence (solid line); transmembrane domain (@); SEC7 region (); putative N-glycosylation sites (*).
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Fig. 2. (Continued ).
classic a/b Rossmann fold and contains a metal iondependent adhesion site (MIDAS) for binding protein ligands (Lee et al., 1995; Qu and Leahy, 1995). The residues (DXSXS) constituting the MIDAS motif in BHS and DS CD11b are completely conserved
and is 100% identical across species compared (Fig. 3). Both BHS and DS contain, 14 predicted N-linked glycosylation sites (Asn-X-Ser/Thr) at identical locations similar to cattle CD11b (Gopinath et al., 2006) as
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Fig. 2. (Continued ).
Table 1 Comparison of CD11b of BHS and DS with that of other species Species/GenBank#
% Identity Bighorn sheep
Cattle (NM 001039957.1) Humans (BC 099660) Chimpanzees (XM 510949) Pigs (U40072) Dogs (XM 843434) Rats (NM 012711) Mice (NM 008401)
Domestic sheep
Nucleotide
Amino acid
Nucleotide
Amino acid
95 88 88 86 87 96 92
92 74 74 80 73 70 70
95 88 88 86 87 96 92
92 74 75 80 73 71 71
The table shows % identity at nucleotide and predicted amino acid levels.
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Fig. 3. Comparison of predicted amino acid sequence of BHS and DS CD11b with that of humans (HuCD11b, BC 099660), cattle (BoCD11b, NM 001039957.1), mice (MoCD11b, NM008401) and rats (RaCD11b, NM012711). The predicted features are indicated by the symbols: I-domain (*); MIDAS motif ($); integrin alpha signature sequence (+); FG-GAP repeats (=); divalent cation binding motif (^).
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Fig. 3. (Continued ).
P.K. Lawrence, S. Srikumaran / Veterinary Immunology and Immunopathology 119 (2007) 287–298 Table 2 Predicted protein characteristics of BHS and DS CD11b
No. of aa Predicted molecular mass (Da) Isoelectric point (pI) Overall charge N-Glycosylation sites Divalent cation binding sites
Bighorn sheep
Domestic sheep
1152 127550.87 7.57 13.5 14 3
1152 127603.95 7.52 13.5 14 3
predicted by NetNGlyc 1.0 server (http:// www.cbs.dtu.dk/services/NetNGlyc/). The BHS and DS CD11b contain seven FG-GAP repeats as seen in cattle, which are predicted to be b propeller structures. There are three potential divalent cation (Ca2+, Mg2+ and Mn2+) binding sites (DX[DN]XDXXXD) which are highly conserved among species and is identical to humans, rats, mice and cattle. BHS and DS CD11b also contain 22 cysteine residues at identical positions, possibly involved in the formation of disulfide bridges. The aa from 63 to 218 are homologous to Saccharomyces cerevisiae SEC7 gene product (Achstetter et al., 1988), which is required for proper protein transport through the Golgi apparatus for post-translational modification. The aa between 343 and 382 contain WD-40 repeats (Neer et al., 1994) which form a circularized beta-propeller structure, necessary for coordinating multi-protein complex assemblies which resonates with the functional properties of integrins. The potential S, T and Y phosphorylation sites are spread all over the CD11b polypeptide, but the cytoplasmic tail contains equal number of S/T phosphorylation sites located at identical positions in both these species. The phylogenetic tree as expected shows a close relationship between BHS and DS CD11b compared to other species (Fig. 4).
Fig. 4. Phylogenetic analysis of CD11b of BHS, DS, and other species. Phylogenetic tree was constructed by neighbor-joining using default settings by online program http://align.genome.jp/sit-bin/clustalw.
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Availability of cDNA for CD11b and CD18 from BHS and DS will enable the development of transfectants expressing recombinant Mac-1 and clarify its role in M. haemolytica-induced cyotoxicity. Acknowledgments This research was supported by funds from the Foundation for North American Wild Sheep and its Eastern, Idaho, Oregon, and Washington Chapters. References Achstetter, T., Franzusoff, A., Field, C., Schekman, R., 1988. SEC7 encodes an unusual, high molecular weight protein required for membrane traffic from the yeast Golgi apparatus. J. Biol. Chem. 263, 11711–11717. Arnaout, M.A., 1990. Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75, 1037–1050. Cywes, C., Godenir, N.L., Hoppe, H.C., Scholle, R.R., Steyn, L.M., Kirsch, R.E., Ehlers, M.R., 1996. Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells. Infect. Immun. 64, 5373–5383. Dassanayake, R.P., Shantalingam, S., Davis, W.C., Srikumaran, S., 2007. Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the b subunit of b2 integrins. Microb. Pathog. (Epub ahead of print). Dassanayake, R.P., Liu, W., Davis, W.C., Foreyt, W.J., Srikumaran, S., submitted for publication. Recombinant expression of Ovis canadensis lymphocyte function-associated antigen-1 in non-susceptible cells identifies it as a receptor for Mannheimia haemolytica leukotoxin. Deshpande, M.S., Ambagala, T.C., Ambagala, A.P., Kehrli Jr., M.E., Srikumaran, S., 2002. Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxininduced cytolysis. Infect. Immun. 70, 5058–5064. Edwards, J.L., Apicella, M.A., 2005. I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells. Cell. Microbiol. 7, 1197–1211. Emsley, J., Cruz, M., Handin, R., Liddington, R., 1998. Crystal structure of the von Willebrand Factor A1 domain and implications for the binding of platelet glycoprotein Ib. J. Biol. Chem. 273, 10396–10401. Forsyth, C.B., Mathews, H.L., 1996. Lymphocytes utilize CD11b/ CD18 for adhesion to Candida albicans. Cell. Immunol. 170, 91–100. Gahmberg, C.G., Valmu, L., Fagerholm, S., Kotovuori, P., Ihanus, E., Tian, L., Pessa-Morokawa, T., 1998. Leukocyte integrins and inflammation. Cell. Mol. Life Sci. 54, 549–555. Gopinath, R.S., Ambagala, A.P.N., Ambagala, T.C., Liu, W., Srikumaran, S., 2006. Molecular cloning and characterization of cDNA encoding CD11b of cattle. Vet. Immunol. Immunopathol. 110, 349–355. Hogg, N., Landis, C.R., Bates, P.A., Stanley, P., Randi, A.M., 1994. The sticking point: how integrins bind to their ligands. Trends Cell Biol. 4, 379–382. Lawrence, P.K., Dassanayake, R.P., Knowles, D.P., Srikumaran, S., 2007. Transfection of non-susceptible cells with Ovis aries
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