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p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1997)

Bottom Line: Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones.The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A.Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Istologia ed Embriologia Generale, Università di Genova, Genova, Italy.

ABSTRACT
Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

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Deglycosylation and  proteolytic digestion of p46 molecules. (a) CD16 (lanes A–D) or p46  (lanes E–H) molecules isolated from  a surface labeled NK cell population  were treated with neuraminidase  (lanes B and F), neuraminidase plus  O-glycanase (lanes C and G), or  N-glycanase (lanes D and H). Untreated molecules are shown in lanes  A and E. Samples were run in an  11% SDS-PAGE under reducing  conditions. (b) p46 and CD16 molecules immunoprecipitated from a  surface labeled NK cell population  were analyzed by SDS-PAGE. Bands  corresponding to p46 (lanes A, C, E,  and G) or CD16 (lanes B, D, F, and H) molecules were excised from the gel and analyzed by SDS-PAGE on a 15–20% gradient gel under reducing conditions after digestion with V8 protease (lanes C and D) or papain (lanes G and H). In lanes A, B, E, and F untreated molecules are shown.
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Figure 2: Deglycosylation and proteolytic digestion of p46 molecules. (a) CD16 (lanes A–D) or p46 (lanes E–H) molecules isolated from a surface labeled NK cell population were treated with neuraminidase (lanes B and F), neuraminidase plus O-glycanase (lanes C and G), or N-glycanase (lanes D and H). Untreated molecules are shown in lanes A and E. Samples were run in an 11% SDS-PAGE under reducing conditions. (b) p46 and CD16 molecules immunoprecipitated from a surface labeled NK cell population were analyzed by SDS-PAGE. Bands corresponding to p46 (lanes A, C, E, and G) or CD16 (lanes B, D, F, and H) molecules were excised from the gel and analyzed by SDS-PAGE on a 15–20% gradient gel under reducing conditions after digestion with V8 protease (lanes C and D) or papain (lanes G and H). In lanes A, B, E, and F untreated molecules are shown.

Mentions: Mice were immunized with the CD3−,16+,56+ NK cell clone SE192 which did not express p58, p70, or p140 NK cell receptor, while it expressed the CD94/NKG2A inhibitory receptor complex identified by the Z199 and Z270 mAbs (11, 12). mAbs were selected on the basis of the ability to modulate the cytolytic activity of the immunizing clone in a redirected killing assay against murine P815 target cells. The selected mAb (BAB281) induced a sharp increase of the cytolytic activity of clone SE192 (Fig. 1 a) as well as that of a panel of NK cell clones expressing one or more NKR. The magnitude of NK cell triggering induced by BAB281 mAb was comparable to that induced by anti-CD16 mAb under the same experimental conditions (Fig. 1 a). On the other hand, a negative control represented by the c218 (isotype-matched, IgG1, anti-CD56) mAb that binds to the NK cell clone, ruled out a nonspecific effect of BAB281 mAb. Surface molecules recognized by BAB281 mAb were analyzed by SDS-PAGE after immunoprecipitation from cell lysates derived from 125I-labeled NK cell populations or clones. As shown in Fig. 1 b, BAB281 mAb immunoprecipitated a single protein of ∼46 kD under both reducing and nonreducing conditions (hereafter termed p46). The comparative analysis of the effect of deglycosylation of p46 or CD16 molecules revealed important differences. As shown in Fig. 2 a, the mobility of p46 molecules was not modified by treatment with N-glycanase, whereas only a slight decrement in molecular weight was induced by neuraminidase followed by O-glycanase treatment. This suggests that, unlike from CD16 (20), p46 molecules do not contain N-linked sugars (whereas O-linked sugars may be present). Further comparative analysis of p46 and CD16 molecules in one-dimensional peptide mapping revealed that the pattern of digestion of the two molecules with either V8 protease or papain was clearly different (Fig. 2 b). Although not shown, the molecular characteristics of p46 molecules were also clearly distinct from other surface molecules mediating NK cell activation, including CD69 and p50. In particular, because of the similar molecular size, p50 and p46 were further compared. First, anti-p46 mAb did not react with cell transfectants expressing one or another member of the p50 family (19, 21); second, p46 molecules did not contain N-linked sugars (Fig. 2 a) whereas p50 molecules did (19, 21). Finally, preclearing experiments on an NK cell clone displaying the p50.3+,p46+ phenotype indicated that after removal of p46 molecules p50.3 molecules could still be immunoprecipitated and vice versa (not shown). Therefore, it appears that BAB281 mAb identifies a novel surface molecule involved in triggering of NK cell–mediated cytotoxicity.


p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1997)

Deglycosylation and  proteolytic digestion of p46 molecules. (a) CD16 (lanes A–D) or p46  (lanes E–H) molecules isolated from  a surface labeled NK cell population  were treated with neuraminidase  (lanes B and F), neuraminidase plus  O-glycanase (lanes C and G), or  N-glycanase (lanes D and H). Untreated molecules are shown in lanes  A and E. Samples were run in an  11% SDS-PAGE under reducing  conditions. (b) p46 and CD16 molecules immunoprecipitated from a  surface labeled NK cell population  were analyzed by SDS-PAGE. Bands  corresponding to p46 (lanes A, C, E,  and G) or CD16 (lanes B, D, F, and H) molecules were excised from the gel and analyzed by SDS-PAGE on a 15–20% gradient gel under reducing conditions after digestion with V8 protease (lanes C and D) or papain (lanes G and H). In lanes A, B, E, and F untreated molecules are shown.
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Related In: Results  -  Collection

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Figure 2: Deglycosylation and proteolytic digestion of p46 molecules. (a) CD16 (lanes A–D) or p46 (lanes E–H) molecules isolated from a surface labeled NK cell population were treated with neuraminidase (lanes B and F), neuraminidase plus O-glycanase (lanes C and G), or N-glycanase (lanes D and H). Untreated molecules are shown in lanes A and E. Samples were run in an 11% SDS-PAGE under reducing conditions. (b) p46 and CD16 molecules immunoprecipitated from a surface labeled NK cell population were analyzed by SDS-PAGE. Bands corresponding to p46 (lanes A, C, E, and G) or CD16 (lanes B, D, F, and H) molecules were excised from the gel and analyzed by SDS-PAGE on a 15–20% gradient gel under reducing conditions after digestion with V8 protease (lanes C and D) or papain (lanes G and H). In lanes A, B, E, and F untreated molecules are shown.
Mentions: Mice were immunized with the CD3−,16+,56+ NK cell clone SE192 which did not express p58, p70, or p140 NK cell receptor, while it expressed the CD94/NKG2A inhibitory receptor complex identified by the Z199 and Z270 mAbs (11, 12). mAbs were selected on the basis of the ability to modulate the cytolytic activity of the immunizing clone in a redirected killing assay against murine P815 target cells. The selected mAb (BAB281) induced a sharp increase of the cytolytic activity of clone SE192 (Fig. 1 a) as well as that of a panel of NK cell clones expressing one or more NKR. The magnitude of NK cell triggering induced by BAB281 mAb was comparable to that induced by anti-CD16 mAb under the same experimental conditions (Fig. 1 a). On the other hand, a negative control represented by the c218 (isotype-matched, IgG1, anti-CD56) mAb that binds to the NK cell clone, ruled out a nonspecific effect of BAB281 mAb. Surface molecules recognized by BAB281 mAb were analyzed by SDS-PAGE after immunoprecipitation from cell lysates derived from 125I-labeled NK cell populations or clones. As shown in Fig. 1 b, BAB281 mAb immunoprecipitated a single protein of ∼46 kD under both reducing and nonreducing conditions (hereafter termed p46). The comparative analysis of the effect of deglycosylation of p46 or CD16 molecules revealed important differences. As shown in Fig. 2 a, the mobility of p46 molecules was not modified by treatment with N-glycanase, whereas only a slight decrement in molecular weight was induced by neuraminidase followed by O-glycanase treatment. This suggests that, unlike from CD16 (20), p46 molecules do not contain N-linked sugars (whereas O-linked sugars may be present). Further comparative analysis of p46 and CD16 molecules in one-dimensional peptide mapping revealed that the pattern of digestion of the two molecules with either V8 protease or papain was clearly different (Fig. 2 b). Although not shown, the molecular characteristics of p46 molecules were also clearly distinct from other surface molecules mediating NK cell activation, including CD69 and p50. In particular, because of the similar molecular size, p50 and p46 were further compared. First, anti-p46 mAb did not react with cell transfectants expressing one or another member of the p50 family (19, 21); second, p46 molecules did not contain N-linked sugars (Fig. 2 a) whereas p50 molecules did (19, 21). Finally, preclearing experiments on an NK cell clone displaying the p50.3+,p46+ phenotype indicated that after removal of p46 molecules p50.3 molecules could still be immunoprecipitated and vice versa (not shown). Therefore, it appears that BAB281 mAb identifies a novel surface molecule involved in triggering of NK cell–mediated cytotoxicity.

Bottom Line: Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones.The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A.Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Istologia ed Embriologia Generale, Università di Genova, Genova, Italy.

ABSTRACT
Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

Show MeSH
Related in: MedlinePlus