Porcine CD58: cDNA cloning and molecular dissection of the porcine CD58–human CD2 interface

The porcine ligands of human CD2 remain unknown in xenotransplantation despite being an important pathway of T cell costimulation. Of the two main candidates, i.e., CD48 and CD58, the cDNA of the most likely ligand poCD58 was cloned from CD48-negative endothelial cells costimulating human CD4þ T cells through the CD2 pathway. The deduced protein sequence is 244 residues long and is 43% homologous to the human sequence. Based on similarity between porcine and human CD58 external V-set Ig-type domains, a structural model of poCD58–huCD2 interaction was built. Most of the charged residues located at the interface with huCD2 are highly conserved. Six putative hydrogen bonds between poCD58 and huCD2 were identified; five involve the same residues as in the syngeneic combination while the sixth is formed between an additional tyrosine in poCD58 and Arg48 in huCD2, increasing the complementarity between the two molecules. These structural data will help us to develop poCD58 blocking agents for xenotransplantation. 2003 Elsevier Inc. All rights reserved.

When pig to human xenotransplantation was con-22 sidered in the early 1990s, the problem of hyperacute 23 rejection was first investigated and recently solved by 24 using organs from pig transgenic for complement regu-25 latory proteins [3]. Cellular rejection phenomena were 26 initially underestimated, mainly because previous mur-27 ine/human xenogeneic experiments suggested that phy-28 logenetic divergence prevented the recognition of ligand/ 29 receptor and receptor/counter-receptor pairs in xenoge-30 neic systems. In particular, human T cell responses to 31 xenogeneic (murine) antigen presenting cells (APCs) 32 were considered low due to inability of costimulatory 33 receptors (CD28, CD2, and LFA-1) to be engaged by 34 the costimulatory molecules on murine APC. However, 35 phylogenetic divergence between pig and human is less 36 than between murine and human. Indeed, strong xeno-37 geneic T cell responses to pig APC were observed and 38 costimulation blocking experiments clearly demon-39 strated that human CD28 [5], CD2, and LFA- 1 [9] could 40 be engaged by the putative porcine counter-receptors. 41 Characterization of porcine costimulatory molecules 42 was therefore initiated with the description of poCD86 43 (also known as B7-2) [7] and then poCD80 [19]. Sub-44 sequently, molecular, functional, and structural char-45 acterization of porcine CTLA4, one of the poCD86 46 ligands, led to the unexpected finding that this molecule 47 bound weakly to human CD86 and CD80 [18] and failed 48 to inhibit human T cell responses, when used as a 49 CTLA4-Ig fusion protein. Porcine ICAM-1 was recently 50 characterized [14] and demonstrated a low degree of 51 conservation with human ICAM-1 (41% identity at the 52 protein level). However, despite being divergent, porcine 53 ICAM-1 has kept the ability to bind human LFA-1 and 54 to transmit costimulatory signals to human T cells [9]. 55 Up to now, in xenogeneic pig-to-primate models, por-56 cine cells have demonstrated their capacity to Biochemical and Biophysical Research Communications 309 (2003)

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57 costimulate human CD4 þ T lymphocytes through the 58 CD2 pathway, but the porcine ligand was not yet 59 identified. Contrary to the situation observed for ro-60 dents (Fig. 1), two distinct ligands, CD58 and CD48, 61 could be engaged by the CD2 T lymphocyte receptor in 62 humans. As CD58 does exist in the ovine species and as 63 it was a ligand of huCD2 [11,12], we hypothesized that 64 huCD2 could be engaged by a porcine ortholog of 65 huCD58. To specifically characterize poCD58, we first 66 searched for a porcine cell line able to costimulate hu-67 man CD4 þ T lymphocyte through the CD2 pathway 68 without expressing poCD48. The porcine L23 lympho-69 blastoid B cell line, which costimulates human T cells by 70 the CD2 pathway [1], was demonstrated to express 71 CD48 (Brossay, A., et al., unpublished) and was dis-72 carded. By contrast, porcine aortic endothelial cells 73 (PAECs), which express a ligand for huCD2 ([9,15], 74 Brossay A et al.,submitted), do not express CD48 75 mRNA (Brossay A et al., submitted) and were therefore 76 selected for CD58 molecular characterization. Porcine 77 CD58 cDNA was entirely cloned from PAEC by a 78 multistep strategy. The amino acid sequence was pre-79 dicted and aligned with the huCD58 protein sequence. 80 Based on the similarity between porcine and human 81 CD58 adhesion domains, a structural model of 82 poCD58-huCD2 interaction is also presented and pro-83 vides insight into the key amino acids in poCD58 that 84 are important for CD2 binding.

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The determined consensual sequence was then used as a probe to 159 screen the porcine EST database from TIGR (The Institute for Ge- Fig. 1. Species-specific differences in the CD2/CD48/CD58 system. In humans and rodents, CD2 and CD244 (2B4) are expressed by lymphocytes (light grey). However in humans, each binds a specific receptor, CD58 and CD48, respectively, presented at the surface of antigen presenting cells or target cells (dark grey). However, huCD48-huCD2 interaction is insufficient to support T-APC cell-cell adhesion (Arulanandam, 1993, JEM). In rodents, the CD48 receptor only is able to bind both CD244 and CD2, and the CD58 gene does not exist. Since CD58 is highly homologous to CD48, it is suspected that the CD58 gene (1p13) arose from CD48 gene (1q21.3-q22) duplication during mammalian phylogenesis, after divergence from rodents.
271 The poCD58 cDNA sequence predicts a 244 residue 272 protein which can be aligned with huCD58 (Fig. 4). As 273 huCD58 has a 28 residue peptide signal sequence, the 274 putative poCD58 signal sequence was supposed simi-275 larly to contain 28 amino acids. As its human counter-276 part, poCD58 is a type I transmembrane protein with an Fig. 2. CD2 pathway blocking experiment. The proliferation of human CD4 þ T cells was tested after 6 days of coculture on irradiated PAEC monolayers, in the presence of blocking agent (anti-CD2b) and nonblocking agent (anti-CD2) at saturating concentrations or their controls. At 18 h before the end of the coculture, wells were pulsed with tritiated thymidine (3.6 Â 10 4 Bq/well) and proliferation was measured by thymidine uptake. Results are expressed in cpm as means AE SD of triplicate determinations. This figure is derived from one experiment representative of five.
A. Brossay et al. / Biochemical and Biophysical Research Communications 309 (2003) (104, 115, 134, 284 140, 163, and 181). Two EST (141404 and 259109) were found that share 100% overlap with the 526 bp fragment. (C) The B58 primers were therefore designed to amplify the entire coding sequence poCD58.2 (912 bp). (D and E) represent poCD58.1 and poCD58.2 band amplification, respectively, in a 1.6% agarose gel with a FX174HaeIII DNA ladder. Fig. 4. Comparison of the amino acid sequence deduced from porcine CD58 cDNA, with human CD58 amino acid sequence. Porcine CD58 sequence shares 43% identity with human CD58 protein. Conserved residues between the two sequences are shaded in grey. N-linked glycosylation sites in human are denoted by a circle and putative N-linked glycosylation sites in pig are denoted by a diamond. The signal peptide sequence is underlined.

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A. Brossay et al. / Biochemical and Biophysical Research Communications 309 (2003) [6]. Indeed, the only conserved N-gly-292 cosylation site between the murine, porcine, and human 293 MHC class I sequences (Asn 86) has been clearly dem-294 onstrated to be involved in the expression and function 295 of a MHC class I antigen [13]. Nonconservation of the 296 three other N-linked glycosylation sites, together with 297 divergence at the peptide levels probably explains why 298 no anti-huCD58 mAbs have been described to cross-299 react with the porcine ortholog. 300 Because the V domain of huCD58 is sufficient for 301 binding to CD2 [8], it is likely that the orthologous V 302 domain is involved in the binding to huCD2 if poCD58 303 proves to be a ligand. Only one amino acid residue in-304 sertion was observed in the porcine sequence, a tyrosine 305 at position 42. This tyrosine insertion is located in the 306 putative C 0 -C 0 0 loop of poCD58, towards the CD2 307 molecule, and probably does not alter the backbone 308 conformation which is likely to be conserved between 309 huCD58 and poCD58. However, this extra amino acid 310 at position 42, together with an extra N-linked glyco-311 sylation site at position 43, could prevent or modify the 312 putative interaction with CD2. 313 Therefore, to gain additional insight into the inter-314 action of poCD58 with huCD2, a structural model of 315 poCD58 V domain was prepared by homology model-316 ing, relying on the high resolution structure of huCD58. 317 The homology-based poCD58 model displayed the same 318 V set Ig-like fold as the huCD58 template (Fig. 4), the 319 rmsd (root-mean-square-deviation) between the two 320 structures being 1.29 A A after superimposing 88 Ca at-321 oms, a value in agreement [4] with the 43% identity 322 between poCD58 and huCD58. The only significant 323 conformational difference between both structures oc-324 curred in the loop between b strands C 0 and C 0 0 due to 325 the insertion of a tyrosyl residue at position 42. 326 The complex between poCD58 and huCD2 was ob-327 tained by superimposing both molecules onto the cor-328 responding components of the X-ray structure of the 329 huCD58-huCD2 complex [20]. Surprisingly, compared 330 to huCD58, most of the charged residues located at the 331 interface between poCD58 and huCD2 are highly con-332 served (Table 1) and could be engaged in salt bridges 333 with complementary charged residues of CD2 (Fig. 5A). 334 This suggests that, as in the human heterophilic com-335 plex, the major source of binding energy would originate 336 from these charged residues. Charge complementarity 337 occurring at the poCD58-huCD2 interface is clearly 338 evidenced in Fig. 5B, which depicts the electrostatic 339 potential surface of huCD2 in the poCD58-huCD2 340 complex. In addition, six hydrogen bonds were identi-341 fied at the interface between poCD58 and huCD2 (Table  342 1). Five of the hydrogen bonds are formed between the 343 same residues as in the human interaction (Table 1) 344 while the sixth is formed between the additional tyrosyl 345 residue 42 of poCD58 and Arg48 of huCD2. Instead of 346 preventing the interaction between poCD58 and 347 huCD2, it is thus likely that this extra amino acid con-348 tributes significantly to the interaction between the two 349 molecules.
350 Amino acid sequence homology with huCD58 was 351 used to model successfully poCD58, and the possible 352 interaction with huCD2 leading to confirm that the 353 cDNA isolated here is the porcine CD58 ortholog. 354 Moreover, conservation of the charged residues 355 involved in the electrostatic interaction and of the hy-356 drogen bonds suggests that poCD58 is the huCD2 li- Corresponding residues in human CD58 forming salt bridges at the huCD58-huCD2 interface (PDB Accession No. 1qa9) are indicated in parenthesis. Fig. 5. Structure of porcine CD58. Ribbon plot superimposition of the porcine CD58 model and of human CD58. The X-ray structure of human CD58 is represented in light grey and the predicted structure of porcine CD58 is shown in dark grey.
A. Brossay et al. / Biochemical and Biophysical Research Communications 309 (2003) 357 gand expressed by porcine endothelial cells and involved 358 in human CD4 þ T lymphocyte costimulation. Devel-359 oping anti-poCD58 specific antibodies would therefore 360 be a promising approach in pig-to-human xenotrans-361 plantation to inhibit human lymphocyte activation 362 triggered by porcine antigen-presenting cells, leaving 363 intact the interactions with human APCs, which are 364 required for anti-infectious defenses. Whether poCD48 365 constitutes another human CD2 ligand remains an open 366 question, notably in porcine cells presenting costimula-367 tory functions and co-expressing CD48 and CD58, such 368 as the L23 lymphoblastoid cell line (see Fig. 6).

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More generally, taking into consideration the obvi-370 ous species-specific differences in the CD2 system be-371 tween human and mice (summarized in Fig. 1) and the 372 still debated phylogenetic history of this system [20][21][22] 373 increasing the knowledge in a third, intermediate spe-374 cies, is of primary importance, and could lead to unex-375 pected finding concerning xenogeneic interactions. 376 While CD244 (2B4) is still totally unknown in the pig 377 species, a cluster of mAb recognizing CD2 has already 378 been defined for several years [10], and a partial putative 379 porcine CD2 cDNA sequence has been recently identi-380 fied (A. Brossay et al. unpublished). Moreover, this 381 family also includes a CD2 homolog which has been 382 found in the genome of the African swine fever virus 383 (ASFV), an infectious agent which specifically infects 384 porcine macrophages and endothelial cells [2,16]. In the 385 context of several putative poCD58 ligands, it is re-386 markable that porcine and ovine CD58 comprise an 387 extra amino acid at position 42 (Tyr and Ser, respec-388 tively), and that this residue in poCD58 could be in-389 volved in electrostatic interactions with its ligands as 390 suggested by our model. Extending the characterization 391 of the porcine ligands of poCD58 and their analysis by 392 molecular modeling would be very useful. Fig. 6. Porcine CD58-human CD2 interface. (A) Stereo view of the key residues of pCD58 (blue) and of hCD2 (yellow) involved in electrostatic interactions or hydrogen bonds across the pCD58-hCD2 interface. The main chain of pCD58 and hCD2 is shown as a ribbon colored in blue and yellow, respectively. (B) pCD58-hCD2 complex showing pCD58 as a blue ribbon and the electrostatic potential surface of hCD2 colored from dark blue (most positive) to red (most negative). Charged residues of pCD58 involved in electrostatic interactions with hCD2 residues are labeled and represented in ball-and-stick format to show the charge complementarity between residues at the interface. These figures were prepared using the program DELPHI (electrostatic potential) and INSIGHT II. (For the interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)