Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications.

Phosphate addition is a posttranslational modification of proteins, and this modification can affect the activity and other properties of intracellular proteins. Different animal species can be used to generate phosphosite-specific antibodies as either polyclonals or monoclonals, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is vital for their use in proteomics and profiling of disease.

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice.

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.

Overview of the generation, validation, and application of phosphosite-specific antibodies.

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Novel multicolor immunofluorescence technique using primary antibodies raised in the same host species.

Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of each secondary antibody with multiple primary antibodies when doing either dual- or triple-labeling immunofluorescence, it is necessary to use primary antibodies raised in different host species such as mouse, rabbit, and goat. However, in many cases, suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the first target protein followed by detection with antiprimary secondary antibody conjugated to NorthernLights™ NL-637 tag (fluorescence in the far-red spectral region). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-493 tag (green fluorescence) and anti-DIG secondary antibody conjugated to a Rhodamine Red X™ tag (red fluorescence). This technique provides good spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have only primary antibodies raised in the same host species at their disposal.

Functional consequences of interactions between human NKR-P1A and its ligand LLT1 expressed on activated dendritic cells and B cells.

Lectin-like transcript-1 (LLT1) (also named osteoclast inhibitory lectin or CLEC2D) is a ligand for the human NKR-P1A (CD161) receptor, present on NK cells and T cells. To further understand the physiological relevance of this interaction, we developed mAbs against LLT1, characterized the expression pattern of LLT1, and explored the functional consequence of LLT1 engagement of the NKR-P1A receptor on NK cells and T cells. LLT1 is expressed on TLR-activated plasmacytoid dendritic, TLR-activated monocyte-derived dendritic cells, and on B cells stimulated through TLR9, surface Ig, or CD40. Interactions between NKR-P1A on NK cells and LLT1 on target cells inhibit NK cell-mediated cytotoxicity and cytokine production and can inhibit TNF-alpha production by TCR-activated NKR-P1A(+) CD8(+) T cells. In contrast, NKR-P1A failed to inhibit or augment the TCR-dependent activation of NKR-P1A-bearing CD4(+) T cells. Expression of LLT1 on activated dendritic cells and B cells suggests that it might regulate the cross-talk between NK cells and APCs.

Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2-C.

NKG2 is a small family of type II transmembrane proteins possessing extracellular C-type lectin domains expressed primarily on NK cells. The function of these proteins is unknown. We have developed mAbs that recognize NKG2 family members on Western blots. Examination of cell extracts from NK cell clones demonstrates that individual NKG2 family members are expressed on subsets of NK cells. Because the anti-NKG2 mAb do not react with the cell surface Ag, signaling function was studied by generating cell lines that express a chimeric receptor consisting of a cytoplasmic and transmembrane domain from NKG2-A or NKG2-C fused to the extracellular segment of mouse NKR-P1C (NK1.1 Ag). Transfectants of the rat NK cell line, RNK-16, were tested for the effect of anti-NK1.1 mAb on cytolytic activity against the FcR+ target cell lines, P388D1 and P815. The anti-NK1.1 mAb stimulated lytic activity and calcium mobilization in the NK cell line expressing the NKG2-C/NKR-P1C chimeric receptor. By contrast, anti-NK1.1 inhibited the lytic activity and failed to stimulate a calcium response in cells expressing the NKG2-A/NKR-P1C chimeric receptor. Immunoprecipitation experiments demonstrated that the inhibitory cytoplasmic tyrosine phosphatase SHP-1 selectively associates with the NKG2-A/NKR-P1C chimeric receptor, but not with the stimulatory NKG2-C/NKR-P1C receptor. These data indicate that NKG2-A and NKG2-C deliver, respectively, inhibitory and activating transmembrane signals in NK cells.

Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits.

CD94 receptors expressed on NK cells have been implicated in the recognition of certain HLA class I allotypes. We now demonstrate that CD94 glycoproteins form disulfide-bonded heterodimers with the NKG2A/B, NKG2C, and NKG2E glycoproteins. NKG2A/B possesses two immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in its cytoplasmic domain, which may be responsible for the inhibitory function of these receptors, whereas other NKG2 proteins lack ITIMs and may potentially transmit positive signals. Structural heterogeneity in the NKG2 gene family and the formation of heterodimers with CD94 provides for the creation of a diverse NK cell repertoire.

Determination of ABO glycosyltransferase genotypes by use of polymerase chain reaction and restriction enzymes.

BACKGROUND: The molecular basis of red cell ABO group antigens has been determined. The genes encoding the group A and B glycosyltransferases and a nonfunctional group O transferase have been cloned and sequenced. All three genes were similar. When compared to the nucleotide sequence of the A gene, the O gene has a one-base deletion that leads to a frame shift and results in a nonfunctional protein. The B gene differs from the A gene at seven nucleotides. STUDY DESIGN AND METHODS: Techniques using polymerase chain reaction and restriction enzymes to determine ABO transferase genotypes from white cell DNA were modified. Nucleotide sequence differences within the genes were analyzed by the application of selected restriction enzymes. Restriction enzymes Asp718 and BstEII were used to analyze the genes at nucleotide 258, and BssHII and Kas I were used to analyze the genes at nucleotide 523. ABO red cell phenotypes were compared in 60 unrelated individuals with ABO transferase genotypes. The ABO phenotypes and genotypes of individuals from two different families were also analyzed to determine if this method could distinguish individuals who were homozygous for A or B transferase genes from those who were heterozygous. RESULTS: The phenotypes and genotypes were consistent for all unrelated individuals, and within the families, heterozygous individuals could be distinguished from homozygous individuals. Nevertheless, two individuals from one family were found to have a group A red cell phenotype, but when the transferase genes were analyzed at nucleotide 523 with enzymes BssHII and Kas I, both A and B transferase genes were detected. Further analysis of the transferase genes at nucleotide 700 by using restriction enzymes Alu I and Hpa II and those at nucleotide 793 by using enzyme BstNI found that both transferase genes in the two individuals were similar to the A transferase gene. CONCLUSION: An A allele of the group A glycosyltransferase was detected that had the same sequence as the B gene at nucleotide 523 but was identical to the A gene at positions 700 and 793. The identification of this variant gene makes genotyping at nucleotide 523 unreliable. However, analysis of the genes at other sites of nucleotide variation may accurately identify phenotypes.

Characterization of a novel gene (NKG7) on human chromosome 19 that is expressed in natural killer cells and T cells.

NKG7 is a cDNA clone generated from a human NK-cell clone. The DNA and predicted aa sequence of NKG7 is not homologous with any previously reported genes or peptides. NKG7 mRNA is expressed in activated T cells and in A-LAK cells isolated from the peripheral blood of normal individuals, and in normal human kidney, liver, lung and pancreas. Furthermore, NKG7 mRNA is expressed at high levels in TCR gamma delta-expressing CTL clones, and in some TCR alpha beta-expressing CTL clones (both CD4+ and CD8+), but is not expressed in other TCR alpha beta-expressing CTL clones and in cell lines representing B cells, monocytes, and myeloid cells. NKG7 mRNA is not expressed in normal human brain, heart, or skeletal muscle. Southern hybridization of NKG7 suggests that NKG7 is a single-copy gene localized to chromosome 19. A hydropathicity profile of the predicted 148 aa polypeptide indicates that NKG7 is a type-I integral membrane protein with a 38-aa extracellular domain and a 61-aa cytoplasmic domain. These results indicate that the NKG7 gene encodes a novel cell surface protein expressed in several cell types, including NK cells and T cells.

A multigene family on human chromosome 12 encodes natural killer-cell lectins.

We previously isolated a series of cDNA clones designated NKG2-A, B, C, and D from a human natural killer (NK) cell library. These transcripts encode a family of type II integral membrane proteins having an extracellular Ca(2+)-dependent lectin domain. The predicted peptides share structural similarities and amino acid sequence similarity with known receptor molecules. In this report, the genomic organization and mRNA expression of each of the genes were studied by using transcript-specific probes. Southern blot experiments reveal that the probes cross-hybridize with a maximum of five genes at high stringency. By probing a Southern blot prepared from a series of hamster/human hybrid somatic cell lines, we demonstrated that all of the hybridizing fragments occur on human chromosome 12. No gene rearrangement and little restriction fragment length polymorphism (RFLP) was observed with these probes. mRNA expression of the NKG2 genes occurred in NK cells and some T cells but not in other hematopoietic cell types or in other tissues tested. Each of the transcripts occurred in all three of the NK cell lines tested: however, the genes were differentially regulated in T cells. NKG2-D was expressed in nine of fourteen T-cell clones or lines in the panel, whereas NKG2-A/B was expressed in three and NKG2-C was expressed in only one. Expression of each of the transcripts was upregulated following T-cell growth factor (TCGF)-induced activation of a cloned NK cell. The limited distribution of these proteins and their sequence similarity with known receptor molecules suggest that they may function as receptors on human NK cells.

Genomic structure of NKG5, a human NK and T cell-specific activation gene.

We reported previously the isolation of a cDNA clone, designated NKG5, encoding a secreted protein that is expressed only in natural killer and T cells and is strongly upregulated upon cell activation. In this report we have isolated the NKG5 gene from a human placental genomic library and sequenced the gene and two kilobases of 5'-flanking DNA. Comparison with the cDNA sequence reveals that the NKG5 gene consists of five exons and four introns. Intron 1 contains a DNA segment that was reported to occur as an exon in 519, a closely related cDNA clone that was isolated from a T-cell library. This result indicates that NKG5 and 519 are alternative splicing products of a single gene. The 5'-flanking region of the NKG5 gene was analyzed for homology with the promoter regions of cytokines and other activation-induced genes showing lymphocyte-specific expression. Several segments displaying sequence similarity were identified. We also identified numerous sequence elements that have strong similarity to known binding sites for transcriptional regulatory proteins including T cell-specific and activation-specific regulatory factors. These findings are consistent with the cell-specific expression and the tight regulatory control that is observed for the NKG5 gene.

Candidate natural killer cell receptors.

Among the high points of immunological discovery has been the identification of antigen-recognizing receptors on B and T cells. Of the lymphocyte populations, only the NK cell receptor remains unknown. Consequently, any newly-recognized, cell-surface molecules expressed selectively on NK cells, especially ones that can transmit a signal to the cell upon appropriate ligand interaction, are possible candidates. This article describes such candidates.

DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells.

We have previously described the isolation of a cDNA clone, designated NKG2, that was expressed in all natural killer (NK) cells tested but not in T or B cells. In the present communication, the original isolate, when used to probe a cDNA library prepared from a CD3- NK cell clone, was found to crosshybridize with a family of transcripts that fell into four distinct groups designated NKG2-A, -B, -C, and -D. Full-length cDNA sequences were determined for each group, and the DNA and inferred peptide sequences were analyzed. All four transcripts encode type II membrane proteins of 215-233 amino acids. NKG2-A and -B peptides appear to be alternative splicing products of a single gene. NKG2-C is highly homologous with group A, having 94% homology in the external (COOH-terminal) domain and 56% homology throughout the internal and transmembrane regions. NKG2-D is distantly but significantly related (21% amino acid homology) to the first three groups. Therefore, NKG2-A, -C, and -D appear to be encoded by distinct genes within a family of NK cell-specific genes. Peptide sequence homology searches demonstrate that the NKG2 peptides are members of a supergene family that includes several other type II membrane proteins. This family is characterized by the presence of a C-type animal lectin domain, and several of its members have demonstrated transmembrane signaling capability.

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes.

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.

A cDNA clone expressed in natural killer and T cells that likely encodes a secreted protein.

We have isolated a series of cross-hybridizing cDNA clones, as a group designated as NKG5, from a human natural killer (NK) cell clone cDNA library. These clones show a high degree of homology with a previously described gene, 519, which was thought to be T cell specific. A comparison of the full-length cDNA sequence of NKG5 and the published sequence of 519 shows that NKG5 lacks a 242-base segment that is found in 519 and that this deletion leads to the use of a different putative translational start codon. Unlike 519, the predicted NKG5 polypeptide has an NH2-terminal sequence that is strongly hydrophobic, characteristic of a signal peptide, and lacks any additional hydrophobic regions in the remainder of the peptide, suggesting that NKG5 encodes a secreted protein. Both NKG5 and 519 are expressed in NK and T cells but not in a variety of other hematopoietic cell lines. NKG5 is an abundant transcript and its level of expression is about 40 times that of 519 in NK and T cells. Southern blot and DNA sequence analyses suggest that NKG5 and 519 mRNAs are transcripts from a single gene that has allelic polymorphism.

Isolation and characterization of NK cell or NK/T cell-specific cDNA clones.

Natural killer (NK) cells are cytotoxic lymphocytes that share numerous cell surface antigens and functional components with T cells. However, in comparison with our knowledge of T cells, little is known about the molecular mechanisms of NK cell activation and function. The following study was initiated as an effort to obtain further information about similarities and differences between NK and T cells at the level of gene expression and also to identify NK-specific cDNA clones for future functional studies of the corresponding gene products. The study used cDNA libraries prepared from an NK clone and from an Epstein-Barr virus transformed B cell lymphoblastoid cell line (LCL). We employed a combination of differential and subtractive hybridization methodologies, which can successfully identify cell-specific cDNA clones representing medium to high abundance transcripts, to identify genes that are expressed in NK cells but not in the LCL. We were particularly interested to ascertain to what extent genes isolated in this manner would be expressed only in NK cells as opposed to being expressed in NK and T cells. Twelve different cross-hybridizing groups were identified that were not expressed in the LCL, and these groups were further characterized: (1) they were used to probe Northern blots prepared from a panel of cells including NK cells, T cells, and B cells: (2) changes in the steady-state level of message following T cell growth factor (TCGF)-induced activation of an NK cell clone were examined for selected isolates; and (3) a partial DNA sequence was determined for each cross-hybridizing group. The DNA sequences of seven groups were identical to previously reported sequences. One group was highly homologous with but not identical to what has been reported as a T cell specific gene, named 519. The DNA sequences of four groups showed no significant homology with the sequences in the GenBank and EMBL databases. The mRNA expression of the newly-identified groups demonstrated several different regulation patterns with respect to cell distribution and level of expression in response to TCGF-activation. Expression of the twelve different genes was examined in three populations of NK cells all of which were CD3- and possessed NK activity. Although these cells differentially expressed the prototype NK markers CD16 and CD56 (the cells were CD16+, CD56-, CD16-, CD56+ and CD16+, CD56+), the expression of all groups of cDNA clones was comparable in the three different types of NK cells despite the phenotypic differences.(ABSTRACT TRUNCATED AT 400 WORDS)