Re-evaluation of Monosaccharide Binding Property of Recombinant Soluble Carbohydrate Recognition Domain of the Natural Killer Cell Receptor NKR-P1A

Abstract

carbohydrate recognition domain As we have been unable to reproduce (1Feizi T. Nature. 1996; 380: 559Google Scholar) the effects of oligosaccharides on killing by natural killer (NK) cells of the rat (2Bezouska K. Yuen C.-T. O'Brien J. Childs R.A. Chai W. Lawson A.M. Drbal K. Fiserova A. Pospisil M. Feizi T. Nature. 1994; 372: 150-157Crossref PubMed Scopus (260) Google Scholar), the need has arisen to re-examine the carbohydrate-binding properties reported for recombinant soluble forms of NKR-P1A, which is a disulfide-linked homodimeric transmembrane protein of lectin type at the surface of NK cells and NK-like T cells, and is an activator of cytotoxicity (3Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (383) Google Scholar, 4Ryan J.C. Niemi E.C. Nakamura M.C. Seaman W.E. J. Exp. Med. 1995; 181: 1911-1915Crossref PubMed Scopus (117) Google Scholar). Attempts to generate the bacterially expressed soluble forms of the monomeric carbohydrate-recognition domain (CRD)1 (5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar) and the dimeric full-length extracellular part (2Bezouska K. Yuen C.-T. O'Brien J. Childs R.A. Chai W. Lawson A.M. Drbal K. Fiserova A. Pospisil M. Feizi T. Nature. 1994; 372: 150-157Crossref PubMed Scopus (260) Google Scholar, 5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar) of the rat NKR-P1A have proven unsuccessful 2K. Bezouska, personal communication.2K. Bezouska, personal communication. in contrast to previously reported data (5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar). We have now used the pET-28a vector (Novagen) to express inEscherichia coli and refold in vitro the His-tagged CRD of NKR-P1A (Ala90–Leu214) containing the six paired cysteines and also the full-length extracellular region (Arg64–Ser223) containing in addition the four putative dimerization cysteines (Fig.1). An attempt to express a third construct, analogous to one reported previously (5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar), spanning Trp115–Leu214, and thus excluding the predicted N-terminal intra-chain disulfide bridge, was abandoned. This is, first, because expression level was too low. Second, by analogy with the recently reported structure of a related protein, CD94 (6Boyington J.C. Riaz A.N. Patamawenu A. Coligan J.E. Brooks A.G. Sun P.D. Immunity. 1999; 10: 75-82Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the deleted region is likely to be an integral part of the CRD of NKR-P1A. In our new approach, there is high level expression both of the CRD and of the full-length extracellular portion of NKR-P1A, and these have been purified from inclusion bodies by nickel affinity chromatography. Refolding by dialysis as described (5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar) yielded no soluble protein. After numerous trials of dilution protocols (7Rudolph R. Lille H. FASEB J. 1996; 10: 49-56Crossref PubMed Scopus (572) Google Scholar) starting with protein at 2–4 mg/ml in 6 m GdnHCl, the full-length extracellular part of NKR-P1A could be obtained in soluble form (about 20% yield) after a 200-fold dilution in 50 mmTris-HCl, pH 8.5, containing 0.8 m arginine and 2 mm Ca2+, and including reduced and oxidized glutathione, 2 and 1 mm, respectively. Under these conditions the protein was in the form of a heterogeneous mixture of monomeric and disulfide-bonded dimeric and aggregated forms (15:5:80 ratio) as assessed by gel filtration analysis and SDS-polyacrylamide gel electrophoresis. As the yield of the dimeric form after anion-exchange chromatography was only 10–20 μg/liter of refolding buffer, which precluded evaluation of folding status, further work is being carried out with the soluble monomeric CRD which, as will be described in detail elsewhere, has been refolded by the dilution approach (7Rudolph R. Lille H. FASEB J. 1996; 10: 49-56Crossref PubMed Scopus (572) Google Scholar), and the folding status corroborated by various techniques, including 1H NMR spectroscopy (see Fig.2). The monomeric CRD has been radioiodinated, and binding has been examined under standard conditions (8Childs R.A. Wright J.R. Ross G.F. Yuen C.-T. Lawson A.M. Chai W. Drickamer K. Feizi T. J. Biol. Chem. 1992; 267: 9972-9979Abstract Full Text PDF PubMed Google Scholar) to the six monosaccharides linked to bovine serum albumin (kindly provided by Dr. Y. C. Lee): fucose20, galactose34, glucose51, mannose35, N-acetylgalactosamine20, and N-acetylglucosamine28, where the subscripts indicate the numbers of monosaccharides per mol of bovine serum albumin. As opposed to a previous report (5Bezouška K. Vlahas G. Horváth O. Jinochová G. Fišerová A. Giorda R. Chambers W.H. Feizi T. Pospı́šil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar), no binding signals to any of the monosaccharides were detected for the CRD. Experiments are in hand to oligomerize the refolded monomeric CRD, which by analogy with selectins (9Galustian C. Childs R.A. Yuen C.-T. Hasegawa A. Kiso M. Lubineau A. Shaw G. Feizi T. Biochemistry. 1997; 36: 5260-5266Crossref PubMed Scopus (42) Google Scholar), is predicted to be a prerequisite for the complete re-evaluation and exploration of the ligands of NKR-P1A. Knowledge of the ligands for this receptor may provide crucial information for understanding how it participates in effector function in nature.