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NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor.

Brooks AG, Posch PE, Scorzelli CJ, Borrego F, Coligan JE - J. Exp. Med. (1997)

Bottom Line: CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s).Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94.Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.

ABSTRACT
CD94 is a C-type lectin expressed by natural killer (NK) cells and a subset of T cells. Blocking studies using anti-CD94 mAbs have suggested that it is a receptor for human leukocyte antigen class I molecules. CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s). This report shows that NKG2A, a 43-kD protein, is covalently associated with CD94 on the surface of NK cells. Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94. Analysis of NK cell clones showed that NKG2A was expressed in all NK cell clones whose CD16-dependent killing was inhibited by cross-linking CD94. The induction of an inhibitory signal is consistent with the presence of two immunoreceptor tyrosine-based inhibitory motifs (V/LXYXXL) on the cytoplasmic domain of NKG2A. Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.

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NKG2A fails to mature in the absence of CD94. (A) Whole cell lysates of NK-92 or YT were either digested with Endoglycosidase Hf (+)  or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (B)  Anti-CD94, anti-NKG2A or control (anti-Ly49), immunoprecipitates from NK-92 cells were either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (C) NK-92 cell  lysates were exhaustively precleared with control (anti-Ly49), anti-CD94 or anti-NKG2A Ab. The remaining lysates were divided in two and either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with antiNKG2A peptide Ab. The immunoblots in B and C were overexposed to detect trace amounts of immature CD94-associated NKG2A in B and to emphasize that no mature NKG2A could be detected in NK-92 lysates after removal of CD94 associated NKG2A in C.
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Figure 3: NKG2A fails to mature in the absence of CD94. (A) Whole cell lysates of NK-92 or YT were either digested with Endoglycosidase Hf (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (B) Anti-CD94, anti-NKG2A or control (anti-Ly49), immunoprecipitates from NK-92 cells were either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (C) NK-92 cell lysates were exhaustively precleared with control (anti-Ly49), anti-CD94 or anti-NKG2A Ab. The remaining lysates were divided in two and either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with antiNKG2A peptide Ab. The immunoblots in B and C were overexposed to detect trace amounts of immature CD94-associated NKG2A in B and to emphasize that no mature NKG2A could be detected in NK-92 lysates after removal of CD94 associated NKG2A in C.

Mentions: The 35-kD NKG2A protein was always detected in whole cell lysates of NKG2A positive cells and is always less abundant than the 40–45-kD form (Fig. 1, also see Fig. 3). The fact that anti-peptide-Ab specific for the amino terminus and mAbs-specific for the lectin domains of NKG2A (data not shown) each react with the 35-kD protein, suggested that it may represent NKG2B. The existence of a putative NKG2B protein was postulated from the detection of transcripts (cDNAs) for an alternative splice variant of NKG2A lacking 54 bases (22). These bases encode 18 amino acids in the stalk or neck region of the protein which links the lectin domain to the transmembrane region and contains a potential N-linked glycosylation site. The absence of this lower molecular mass form in anti-CD94 immunoprecipitates suggests that it lacks the structural requirements for pairing with CD94.


NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor.

Brooks AG, Posch PE, Scorzelli CJ, Borrego F, Coligan JE - J. Exp. Med. (1997)

NKG2A fails to mature in the absence of CD94. (A) Whole cell lysates of NK-92 or YT were either digested with Endoglycosidase Hf (+)  or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (B)  Anti-CD94, anti-NKG2A or control (anti-Ly49), immunoprecipitates from NK-92 cells were either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (C) NK-92 cell  lysates were exhaustively precleared with control (anti-Ly49), anti-CD94 or anti-NKG2A Ab. The remaining lysates were divided in two and either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with antiNKG2A peptide Ab. The immunoblots in B and C were overexposed to detect trace amounts of immature CD94-associated NKG2A in B and to emphasize that no mature NKG2A could be detected in NK-92 lysates after removal of CD94 associated NKG2A in C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196137&req=5

Figure 3: NKG2A fails to mature in the absence of CD94. (A) Whole cell lysates of NK-92 or YT were either digested with Endoglycosidase Hf (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (B) Anti-CD94, anti-NKG2A or control (anti-Ly49), immunoprecipitates from NK-92 cells were either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with anti-NKG2A peptide-specific Ab. (C) NK-92 cell lysates were exhaustively precleared with control (anti-Ly49), anti-CD94 or anti-NKG2A Ab. The remaining lysates were divided in two and either digested with Endoglycosidase Hf, (+) or mock digested (−), separated by SDS-PAGE under reducing conditions and analyzed by Western blot with antiNKG2A peptide Ab. The immunoblots in B and C were overexposed to detect trace amounts of immature CD94-associated NKG2A in B and to emphasize that no mature NKG2A could be detected in NK-92 lysates after removal of CD94 associated NKG2A in C.
Mentions: The 35-kD NKG2A protein was always detected in whole cell lysates of NKG2A positive cells and is always less abundant than the 40–45-kD form (Fig. 1, also see Fig. 3). The fact that anti-peptide-Ab specific for the amino terminus and mAbs-specific for the lectin domains of NKG2A (data not shown) each react with the 35-kD protein, suggested that it may represent NKG2B. The existence of a putative NKG2B protein was postulated from the detection of transcripts (cDNAs) for an alternative splice variant of NKG2A lacking 54 bases (22). These bases encode 18 amino acids in the stalk or neck region of the protein which links the lectin domain to the transmembrane region and contains a potential N-linked glycosylation site. The absence of this lower molecular mass form in anti-CD94 immunoprecipitates suggests that it lacks the structural requirements for pairing with CD94.

Bottom Line: CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s).Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94.Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.

ABSTRACT
CD94 is a C-type lectin expressed by natural killer (NK) cells and a subset of T cells. Blocking studies using anti-CD94 mAbs have suggested that it is a receptor for human leukocyte antigen class I molecules. CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s). This report shows that NKG2A, a 43-kD protein, is covalently associated with CD94 on the surface of NK cells. Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94. Analysis of NK cell clones showed that NKG2A was expressed in all NK cell clones whose CD16-dependent killing was inhibited by cross-linking CD94. The induction of an inhibitory signal is consistent with the presence of two immunoreceptor tyrosine-based inhibitory motifs (V/LXYXXL) on the cytoplasmic domain of NKG2A. Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.

Show MeSH
Related in: MedlinePlus