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J Biol Chem, Vol. 274, Issue 42, 30335-30335, October 15, 1999
From The Glycosciences Laboratory, Imperial College School of
Medicine, Northwick Park Campus, Harrow HA1 3UJ, United Kingdom
As we have been unable to reproduce (1) the
effects of oligosaccharides on killing by natural killer (NK) cells of
the rat (2), 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 (3, 4). Attempts to generate the bacterially expressed
soluble forms of the monomeric carbohydrate-recognition domain
(CRD)1 (5) and the dimeric
full-length extracellular part (2, 5) of the rat NKR-P1A have proven
unsuccessful2 in contrast to
previously reported data (5).
We have now used the pET-28a vector (Novagen) to express in
Escherichia 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 (5), 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 (6),
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 (5) yielded no
soluble protein. After numerous trials of dilution protocols (7)
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 mM
Tris-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 (7), and the folding status corroborated by various
techniques, including 1H NMR spectroscopy (see Fig.
2).
Re-evaluation
of Monosaccharide Binding Property
of Recombinant Soluble Carbohydrate Recognition Domain of the Natural
Killer Cell Receptor NKR-P1A*
,
View larger version (9K):
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Fig. 1.
Schematic diagram of NKR-P1A of the rat,
depicting the methionine (M1) at the
cytoplasmic N terminus, the transmembrane segment
(TM), three predicted intramolecular disulfide
bridges, and the four unpaired cysteines, which may be involved in
dimerization. Also shown are the limits of the three recombinant
constructs: the CRD (A90-L214),
the full-length extracellular region
(R64-S223), and the truncated
CRD region (W115-L214).
View larger version (18K):
[in a new window]
Fig. 2.
1H NMR spectrum at 600 MHz of the
refolded monomeric CRD. Approximately 300 µM was
analyzed in D2O at pH 8.3, 22 °C.
The monomeric CRD has been radioiodinated, and binding has been examined under standard conditions (8) 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 (5), 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 (9), 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.
 |
FOOTNOTES |
|---|
Re-evaluation of a paper entitled "Rat
natural killer cell antigen, NKR-PI, related to C-type animal lectins
is a carbohydrate-binding protein" published in Vol. 269 (1994)
16945-16952 by K. Bezouska, G. Vlahas, O. Horvath, G. Jinochova, A. Fiserova, R. Giorda, W. H. Chambers, T. Feizi, and M. Pospisil.
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Unite de chimie Organique, Institut
Pasteur, Paris 75724 Cedex 15, France.
§ To whom correspondence should be addressed. Tel.: 44-181-869-3460/3461; Fax: 44-181-869-3455; E-mail: t.feizi@ic.ac.uk.
2 K. Bezouska, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviation used is: CRD, carbohydrate recognition domain.
| |
REFERENCES |
|---|
| 1. | Feizi, T. (1996) Nature 380, 559 |
| 2. | Bezouska, K., Yuen, C.-T., O'Brien, J., Childs, R. A., Chai, W., Lawson, A. M., Drbal, K., Fiserova, A., Pospisil, M., and Feizi, T. (1994) Nature 372, 150-157[CrossRef][Medline] [Order article via Infotrieve] |
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| 6. | Boyington, J. C., Riaz, A. N., Patamawenu, A., Coligan, J. E., Brooks, A. G., and Sun, P. D. (1999) Immunity 10, 75-82[CrossRef][Medline] [Order article via Infotrieve] |
| 7. | Rudolph, R., and Lille, H. (1996) FASEB J. 10, 49-56[Abstract] |
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Childs, R. A.,
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| 9. | Galustian, C., Childs, R. A., Yuen, C.-T., Hasegawa, A., Kiso, M., Lubineau, A., Shaw, G., and Feizi, T. (1997) Biochemistry 36, 5260-5266[CrossRef][Medline] [Order article via Infotrieve] |
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