Identification of CD226 ligand on colo205 cell surface

Abstract

AIM: To confirm the existence of CD226 ligand and its distribution, which is a novel molecule that was cloned in 1996.

METHODS: The mRNA was extracted from TPA activated Jurkat cells and used as a template for reverse-transcription. After PCR amplification, the fragment including CD226 extracellular region and the splice donor sequence “ACTTACCTGT” was obtained and cloned into fusion expression vector pIG. The recombinant vector pCD226/Ig was transfected in COS-7 cells by DEAE-Dextran method, the secreting fusion protein was identified by Sandwich ELISA, and was purified by anti-CD226 affinity chromatography. This fusion protein was used as a probe in the investigation of CD226 ligand by immunohistochemistry. Existence of CD226 ligand was further identified by adhesion experiment.

RESULTS: Expression of a secreting fusion protein was identified by sandwich ELISA, indicating that both CD226 extracellular domain and IgGFc domain could be recognized respectively by anti-CD226 and anti-hIgFc mAb. About 130 μg CD226/Ig fusion protein could be obtained from 100 mL COS-7 culture supernatants by anti-CD226 affinity chromatography purification. SDS-PAGE showed that this fusion protein has a molecular mass of 83 ku. It was confirmed by immunohistochemistry that CD226 ligand expressed on the Colo205 cells, but not on Jurkat cell, U937 cell and mixed lymphocyte culture cells. In adhesive assay, resting Jurkat cells did not have significant adhesion to Colo205 cells. In contrast, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesive reaction could be blocked by CD226/Ig fusion protein or anti-CD226 mAb. Immunochemical experiment showed that Colo205 cells could be specifically stained by CD226/Ig, indicating that CD226 ligand exists on the surface of Colo205 cells.

CONCLUSION: Existence of CD226 ligand on the surface of Colo205 cells was identified by immunohistochemistry and adhesion blocking experiment. In addition, the secreting CD226/Ig fusion protein prepared in this study will be a potential tool for further investigation of CD226 ligand.

Key Words: $[Keywords]

INTRODUCTION

CD226 was initially reported by Burns in 1985 with a name TLiSA1 (T lineage specific activation antigen 1, TLiSA1)[1], and was renamed PTA1 (platelet and T cell antigen 1, PTA1) in 1989 because of its expression on platelet as well[2]. Since 1985, a lot of investigations have been done on CD226 expression, functions and its relationship with diseases[2-6]. CD226 was mainly expressed on the activated T cells, NK cells, monocytes, platelets and megakaryocytes lineage[4-8], taking part in signal transduction of T cell activation and differentiation as well as platelet activation and aggregation. CD226 mAb was found to stimulate the activation and aggregation of platelets[2,3], inhibit differentiation of CTL[2,9] and T and NK cell mediated cytotoxicity[7]. In 1997, PTA1 cDNA was cloned from cDNA library of TPA activated Jurkat cells[10]. It belongs to an immunolglobulin super family (IgSF) with a 232aa extracellular region, 25aa transmembrane region and 61aa intracellular region. Interestingly, PTA1 cDNA was almost the same as that of DNAM-1 (DNAX associated molecule-1), a novel molecule cloned by Shibuya in 1996[7]. In 2000, PTA1 (DNAM-1) was designated as CD226 on the 7th Workshop and Conference on the Human Leucocyte Differentiation Antigen in Harrogate. CD226 is the only IgSF member whose extracellular region consists of two IgV-like domains.

Almost all the important Ig-superfamily members are highly conserved during molecular evolution[11-13], such as CD2, CD4/ CD8 and CD28. Homological analysis showed that CD226 molecules are highly conserved (93%-95%) among human, ape and monkey, suggesting that CD226 may have very important functions[14]. It seems that CD226 takes part in the mechanism of platelet function disorders, autoimmune diseases, transplantation, virus infection diseases and tumors[3,15-20], suggesting that CD226 plays an important role in immune system and may have a potential application to clinical diagnosis and treatment. Up to, little is known about CD226 ligand. So, the aim of this study is to make a preliminary research on CD226 ligand.

MATERIAL AND METHODS

Materials

pIG vector was a gift kindly presented by Dr. Xu. COS-7, U937, Jurkat, Colo205 cell line and CD226 hybridoma (secreting mAb Leo-A1 against the extracellular domain of CD226) were provided by Prof. Jin. Goat anti-human Ig-SABC immunohistochemistry kit was purchased from Boster Co. Vectors and restrict enzymes were purchased from Huamei Co; Trizol reagent kit was purchased from Gibco Co, and CNBr-Sepharose 4B is a product of Pharmacia Co.

Design of the primers for semi-nest RT-PCR

Primers were designed according to the sequence of CD226 cDNA: primer 1, 5’-GCAAGCTTCAGAGATGGATTATCCTACT-3’ (forward, containing HindIII enzyme site at 5’); primer 2, 5’-GCGGATCCACTTACCTGTAGCCACAAAGAGGGTATATTGG-3’ (reverse, containing BamHIenzyme site and donor splice region “ACTTACCTGT” at 5’); primer 3, 5’-ACTCTAGTCTTTGGTCCTGC-3’. Primer1 and Primer 3 were used to amplify the whole length CD226 cDNA, and primer 1 and primer 2 were used to amplify cDNA encoding CD226 extracellular region (Figure 1).

Figure 1 Sequence of CD226 cDNA and primers. 174-923 bp: extracellular region of CD226; 174-227 bp: signal peptide sequence; 924-998 bp: transmembrane region; labeled sequence: primers; under-lined sequence was the splicing donor sequence.

Reverse transcription-polymerase chain reaction (RT-PCR)

Jurkat cells were cultured at a concentration of 1 × 10 ⁹·L^-1 with TPA (50 μg·L^-1) for 18-30 h at 37 °C, and the total RNA was isolated by Trizol reagent kit. RNA was primed and reverse transcribed into cDNA in a 20 μL reaction volume containing 10U reverse transcriptase, 10 μg RNA, 10 × Buffer 2 μL, 10 mmol·L^-1 dNTP complex 4 μL, and primer 3. After incubation at 42 °C for 1 h, the reverse transcription product was PCR amplified by Primer 1 and Primer 3, and then by Primer 1 and Primer 2. The PCR parameters were 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 80 s for 35 cycles.

Plasmid construction

Expression vector for CD226 extracellular region was prepared as previously described[21]. The corresponding PCR fragment was cloned into pUC-19 and M13mp18/19 vector for DNA sequencing. After DNA sequencing, the 793 bp fragment encoding CD226 extracellular region and the splice donor sequence “ACTTACCTGT” was subcloned into pIG, a mammalian fusion protein expression vector containing the cytomegalovirus (CMV) promoter, splice acceptor, genomic human IgG1, and SV40 polyadenylation signal. The resulting expression vector pCD226/ Ig was identified by PCR and restrict enzyme digestion.

COS-7 cell Transfection

COS-7 cell transfection was performed by a modified method[22]. Briefly, COS-7 cells at 50%-75% confluence were transfected in 7 mL glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 100 mL·L^-1 fetal calf serum (FCS), 400 μg DEAE - dextran/mL, 100 μmol·L^{- 1} chloroquine diphosphate, and 40 μg purified DNA. After 3 h at 37 °C, the transfection mixture was removed and the cells were treated with 100 g·L^-1 dimethyl sulfoxide in PBS for 2 min. Cells were then returned to DMEM supplemented with 10 mL·L^-1 fetal calf serum (FCS) for 4 d to allow CD226/Ig expression.

Purification and identification of CD226/Ig fusion protein

Leo-A1 affinity chromatography column was prepared as previously described[23]. The pCD226/Ig transfected COS-7 supernatant was spined out cell debris for 5 min at 10000 r·min^-1, filtered through a 0.45 μm filter and then purified by Leo-A1 affinity chromatography. The expression of a secreting fusion protein was identified by sandwich ELISA (anti-CD226 mAb and HRP-conjugated anti-hIg mAb, anti-IL-8 antibody was used as control). SDS-PAGE and Western-blot (polyclonal anti-hIgG antibody) were performed by routine methods.

Immunohistochemical staining by CD226/Ig

Immunohistochemical experiment was performed by SABC methods. For light microscopy, cytospin preparations of Colo205 cells were incubated with 3 g·L^-1 methanol-hydrogen peroxide solution at 37 °C for 30 min to inactivate endogenous peroxidases. The slides were then blocked in goat serum blocking solution at 37 °C for 30 min. Without washing, the slides were incubated with CD226/Ig fusion protein (10 mg·L^-1) at 4 °C. After overnight incubation, the slides were washed three times with PBS, and incubated with biotin labeled goat anti human IgG for 4 h at room temperature. Finally, the slides were washed, allowed to sit for 2 min, and then reacted with SABC complex and DAB peroxidase substrate solution. Human IgG1 was used instead of CD226/Ig in the control experiment.

Adhesion assay

In 96 well plate, Colo205 cells with the density of 0.5 × 10⁵/well were cultured for one day to let cells confluence, and 3.7 MBq ⁵¹Cr labeled resting or activated Jurkat cells were added with 1 × 10⁵/well and cultured for 4 h, and non-adhered cells were moved by gently washing, the adhered cells were lysed by 20 g·L^-1 Triton X-100, and Bq was measured. Different concentrations of CD226/Ig fusion protein and anti-CD226 mAb Leo-A1 were used in the adhesion blocking experiment

RESULTS

Construction of pCD226/Ig fusion expression vector

A very low level of CD226 molecules was expressed on resting Jurkat cells, and the expression level could be enhanced by TPA stimulation for 24 h. The CD226 mRNA from Jurkat cells was enriched by TPA stimulating. After retro-transcription, semi-nests PCR amplification, a single band about 800 bp has been obtained. This fragment was then ligated into pUC19 and subcloned into M13mp18/19 vector for sequencing. The sequencing data showed that the fragment was 793 bp long in total, a HindIII enzyme site could be found at the upstream and the “ACTTACCTGT”donor splicing sequence and a BamHI site could be found at the downstream. The 793 bp fragment was ligated between HindIII and BamHI sites of pIG vector. The recombinant vector pCD226/ Ig was identified by restrict enzyme digestion assay and PCR identification (Figure 2).

Figure 2 Construction of pCD226/Ig fusion vector.

Expression, identification and purification of CD226/Ig fusion protein

The pCD226/Ig vector was transiently transfected into COS-7 cells. The optimal conditions was checked before large-scale transfection, and it was found that transfected COS-7 cells cultured in medium with 10 mL·L^-1 serum for 96 h could produce CD226/Ig most effectively. The secreted CD226/Ig fusion protein was purified by Leo-A1 affinity chromatography, About 130 μg CD226/Ig fusion protein could be obtained from 100 mL COS-7 culture supernatants by anti-CD226 affinity chromatography purification. SDS-PAGE showed CD226/Ig fusion protein has a molecular mass of 83 ku (Figure 3) and could be recognized by anti-IgG polyclonal antibody in Western-blot assay (Figure 4). In ELISA assay, CD226/Ig expression could be identified in pCD226/Ig transfected COS-7 supernatant, but a negative result was obtained in pIG vector transfected COS-7 supernatant (Table 1). Both CD226 extracellular domain and Fc domain of the CD226/Ig protein could be recognized respectively by anti-CD226 and anti-hIg Fc mAb, suggesting that CD226/Ig could mimic the nature CD226 molecule and be a potential tool in the research of CD226 ligand and its functions.

Table 1 Identification of CD226/Ig fusion protein expression by sandwich-ELISA.

Coated antibody	Samples tested	Conjugated secondary antibody	Results
Anti-CD226	pCD226/Ig transfected COS supernatant	HRP-Gα hIgG	+++
Anti-CD226	pIG transfected COS supernatant	HRP-Gα hIgG	-
Anti-IL-8	pCD226/Ig transfected COS supernatant	HRP-Gα hIgG	-

Figure 3 Identification of CD226/Ig fusion protein purified by Leo-A1 Sepharose-4B affinity column. 1. Supernatant of pIG vector transfected COS-7 cells; 2. Supernatant of pCD226/Ig transfected COS-7 cells; 3. Supernatant of pCD226/Ig trans-fected COS-7 cells after concentrated by ammonium sulfate; 4. Flow through solution of pCD226/Ig transfected COS-7 cells after affinity chromatography; 5. CD226/Ig fusion protein purified by Leo-A1 sepharose-4B affinity column; 6. Marker.

Figure 4 Western-blot result of CD226/Ig by anti-IgG polyclonal antibody Lane 1. Supernatant of pCD226/Ig transfected COS-7 cells; Lane 2. Super-natant of pIG transfected COS-7 cells

Identification of CD226 ligand on Colo205 cells

Immunohistochemical experiment showed that Colo205 cells could be specifically stained by CD226/Ig but not by control protein (hIgG), indicating that CD226 ligand exists on the surface of Colo205 cells (Figure 5). Resting Jurkat cells had a low-level expression of CD226 on cell surface, whereas activated Jurkat cells expressed high level of CD226 after TPA treatment for 24 h. In adhesive assay, resting Jurkat cells have a very low level adhesion to Colo205 cells. In contrast, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesive reaction could be blocked by CD226/Ig fusion protein or Leo-A1 mAb but not control Ig and anti-IL-1 mAb, indicating that CD226 takes part in this specific adhesion (Figure 6).

Figure 5 CD226L expression on Colo205 cells identified by CD226/Ig immunohistochemistry. A: Colo205 negative control (stained with hIg); B: Colo205 cells were specifically stained by CD226/Ig.

Figure 6 Adhesion experiments of activated Jurkat cells with Colo205 cells. Density of Colo205 was 0.5 × 10⁵ per well. Density of Jurkat was 1 × 10⁵ per well. R stand for resting Jurkat cells. A stand for activated Jurkat cells.

DISCUSSION

CD226 is a 65 ku glycoprotein expressed on the surface of activated T cells, NK cells, and platelets. It is a member of the Ig-superfamily containing 2 Ig-like domains of the V-set and is encoded by a gene on human chromosome 18q22.3[24]. Ig-superfamily members play essential roles in many aspects of immune responses by acting as immunoglobulin Fc receptors, cytokine receptors, adhesive molecules and accessory molecules[11,25-27]. The main function of Ig-super family concentrated in cell-to-cell recognition and interaction, such as LFA-1/CD2, CD4/MHC, CD28/B7, and many of these Ig-super family members are involved in the T cell activation[28-32]. Cross-linking CD226 with antibodies cause the activation of T cells and aggregation of platelets. Previous studies also showed the cross-linking of LFA-1 induced tyrosine phosphorylation of CD226, in which the Fyn protein tyrosine kinase may play a role[33,34]. The above results indicate that CD226 mediates cellular adhesion to other cells bearing an unidentified ligand and takes part in signal transduction. Interaction between ligand and receptor is the basis of immune response. So, identification of CD226L is very important for the further investigation of CD226.

Intercellular adhesive molecules play an important role in the immune response, they provide not only intercellular binding, but also participate in signal transduction, and are closely related with allograft rejection, tolerance, cell differentiation, lymphocyte homing and tumor immunity[35-39]. In addition to the specific antigen recognition signal provided by CD3-TCR complex, a lot of other signals are required in the T cells mediated immune response, such as signals provided by CD4/CD8, CD2, LFA-1, CD28[40-42]. Our previous results showed that resting T cells expressed low-level of CD226 molecule. When T cells were activated, they expressed high-level CD226 molecules. This finding suggested that CD226 might be closely related to the function of activated T cells. Differentiation of T cell to CTL could be inhibited when Leo-A1 mAb or Leo-A1 F(ab’)₂ were added into mixed lymphocyte culture (MLC), and production of LAK cells was also reduced, this action of Loe-A1 was not dependent on Fc of Leo-A1. The inhibiting effect of Loe-A1 only works in the differentiation phase of CTL but not in the cytotoxicity phase, suggesting that the epitope recognized by CD226 is related with the differentiation of CTL[1,2,7]. Another mAb DX11 against CD226 (DNAM-1) was reported to significantly inhibit the cytotoxicity of CTL to several tumor cell lines, such as Colo205, PA-1, MCF7[7]. Therefore, epitope recognized by DX11 mAb was different from that recognized by Leo-A1 mAb, and these two different epitopes may take part in the differentiation and cytotoxicity of CTL.

Since the mid 1980s, there has been a rapid increase in our knowledge about the specific cell surface molecules mediating cell-cell interaction and adhesion events. This has been largely due to the success of molecular cloning techniques, allowing the isolation of functional cDNA clones that encoding these glycoproteins. Always methods have been successfully used in the cloning of novel cDNA. One of these methods reported by Aruffo and Seed is very effective[43,44]. This method is based on the transient expression of cDNA library in cells and specific mAb recognition (capture and panning). Several molecules, such as CD2, CD22, CD28, CD36 and CD58[43-47], have been cloned by this method. So, once a suitable antibody, ligand or cell line has been identified to recognize a cell surface molecule, the cDNA encoding this molecule could be cloned by this method. CD226 molecule or its homologue to be used in the investigation of CD226 ligand should have the following characteristics: easy to obtain, high in purity and natural in motif. So these molecules could be obtained by purification from platelet, preparations of anti-idiotype mAb or preparations of recombinant CD226 molecule. In this study, we prepared the CD226/Ig fusion protein containing CD226 extracellular region 232aa (including 10 glycosylation sites, 57 ku) and IgG Fc region (CH2, CH3 and H region, 26 ku), the putative molecule weight is consistent with that identified in SDS-PAGE and Western-blot. CD226/Ig fusion protein is expressed in secreting form that was detected by Sandwich-ELISA, its two different domains could be recognized by anti-CD226 mAb and anti-hIg Fc mAb, respectively, but could not be recognized by other mAb, suggesting CD226/Ig can mimic the nature of CD226 molecule and can be used in the research of CD226 ligand. Ig fusion proteins have many advantages such as easy to purify, easy to label and easy to detect, and thus were widely used in ligand identification[48-53], and disease protection[54-56]. These advantages make it more convenient to further clone CD226 ligand.

Immunohistochemical research suggested that CD226 ligand was expressed on Colo205 cells and this result was also supported by adhesion-blocking experiment. It was shown that resting Jurkat cells expressed very low level of CD226 molecules, but activated Jurkat cells expressed high level CD226 molecules after TPA activation for 24 h, and the expression of CD226 was regulated by cytokines, such as TGF-β, TNF-α and IL-2[57]. In the experiment about adhesion, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesion reaction could be blocked by CD226/Ig fusion protein or Leo-A1 mAb but not by control Ig and anti-IL-1 mAb, indicating that CD226 ligand exist in Colo205 cells and takes part in this specific adhesion.

When T cells were activated, they expressed high-level CD226 molecules. Interestingly, CD226 ligand seemed to express on the surface of tumor cells. Activated T cells not only need the specific antigen recognition by TCR, but also the engagement of accessory molecules on T cells by their respective ligans expressed on the target cells[38,39,58,59]. More researches need to be done to reveal their relationship and what signal have CD226 transducted during immune response against tumors. The above results made a solid foundation for further cloning of CD226 ligand and is helpful for thorough investigations on CD226L structure and function.

ACKNOWLEDGMENTS

Dr. Xu and Dr. Lan for their valuable technical advice and assistance.