A Potential Role of Myeloid DAP12-Associating Lectin (MDL)-1 in the Regulation of Inflammation in Rheumatoid Arthritis Patients

The pathogenic roles of myeloid DAP12-associating lectin-1(MDL-1) and DAP12 in human rheumatoid arthritis (RA) remain unknown. Frequencies of MDL-1-expressing monocytes in 22 active RA patients, 16 inactive RA patients, 12 osteoarthritis (OA) patients and 10 healthy controls (HC) were determined by flow-cytometry analysis. The mRNA expression levels of MDL-1 and DAP12 on PBMCs were evaluated by quantitative PCR, and their protein expression levels in the synovium were examined by immunohistochemistry. Significantly higher median percentages of circulating MDL-1-expressing monocytes were observed in active RA patients (53.6%) compared to inactive RA patients (34.1%), OA patients (27.9%), and HC (21.2%). Levels of MDL-1 and DAP12 gene expression in PBMCs and their protein expression in the synovium were significantly higher in active RA patients than in inactive RA or OA patients. MDL-1 levels were positively correlated with parameters of disease activity, articular damage, and levels of proinflammatory cytokines. MDL-1 activator (Dengue virus type 2 antigen) stimulation on PBMCs resulted in significantly enhanced levels of proinflammatory cytokines in RA patients compared to those in OA patients or HC, indicating that MDL-1 activation is functional. Frequencies of MDL-1-expressing monocytes and levels of MDL-1 and DAP12 gene expression significantly decreased after effective therapy. Concordant overexpression of MDL-1 and DAP12 were correlated with increased production of proinflammatory cytokines in RA patients, suggesting their roles in regulating articular inflammation.     

The role of the DAP12 signal in mouse myeloid differentiation

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.     

NKG2D splice variants: a reexamination of adaptor molecule associations

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naïve NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naïve NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.     

Cutting edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells

Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRP beta 1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRP beta 1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRP beta 1/DAP12 complex formation was required for efficient cell-surface expression of SIRP beta 1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRP beta 1 is a new DAP12-associated receptor involved in the activation of myeloid cells.     

Cutting edge: inhibition of TLR and FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12

DAP12 is an ITAM-containing adapter that associates with receptors in myeloid and NK cells. DAP12-associated receptors can give activation signals leading to cytokine production; however, in some situations, DAP12 inhibits cytokine production stimulated through TLRs and FcRs. Here we show that Triggering Receptor Expressed on Myeloid cells (TREM)-2 is responsible for the DAP12-mediated inhibition in mouse macrophages. A chimeric receptor composed of the extracellular domain of TREM-2 and the cytoplasmic domain of DAP12 inhibited the TLR- and FcR-induced TNF production of DAP12-deficient macrophages, whereas a TREM-1 chimera did not. In wild-type macrophages, TREM-2 knockdown increased TLR-induced TNF production. A TREM-2 Fc fusion protein bound to macrophages, indicating that macrophages express a TREM-2 ligand. Thus, the interaction of TREM-2 and its ligand results in an inhibitory signal that can reduce the inflammatory response.     

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1.

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.     

Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells

We have identified and characterized two mouse cDNAs in a mouse antigen-stimulated bone marrow-derived mast cell cDNA library, both of which encode type I transmembrane proteins. The genes were closely mapped in the distal region of mouse chromosome 11 and expressed not only in mast cells but also widely in leukocytes. The extracellular domains of their encoded proteins contain a single variable immunoglobulin (Ig) motif sharing about 90% identity with amino acids, showing that they comprise a pair of molecules and belong to the Ig superfamily. We named these molecules leukocyte mono-Ig-like receptor1 and 2 (LMIR1 and 2). The intracellular domain of LMIR1 contains several immunoreceptor tyrosine-based inhibition motifs (ITIMs). When cross-linked, the intracellular domain was tyrosine phosphorylated and capable of recruiting tyrosine phosphatases, SHP-1 and SHP-2 and inositol polyphosphate 5-phosphatase, SHIP. LMIR2, on the other hand, contains a short cytoplasmic tail and a characteristic transmembrane domain carrying two positively charged amino acids associated with three kinds of immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecules, DAP10, DAP12, and FcRgamma. These findings suggest that a new pair of ITIM/ITAM-bearing receptors, LMIR1 and 2, regulate mast cell-mediated inflammatory responses through yet to be defined ligand(s).     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

The sialate-pyruvate lyase from pig kidney. Elucidation of the primary structure and expression of recombinant enzyme activity.

The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.     

Feline cytokines TNFα AND IL‐1β: PCR cloning and sequencing of cDNA

Abstract

Using oligonucleotide primers and polymerase chain reaction (PCR), cDNAs for feline cytokines TNFα and IL‐1β were amplified, cloned, and sequenced. The cDNA for PCR amplification was prepared from mRNA derived from lipopolysaccharide (LPS) stimulated feline bone marrow derived macrophages. PCR was performed using sets of oligonucleotide primers designed to specifically amplify cDNAs for IL‐1β or TNFα. PCR fragments were cloned into pGEM 3ZF(‐) or pCR 1000 vectors, sequenced and consensus nucleotide sequences reported.

The cDNA for feline TNFα had a 98.6% match with coding regions of a genomic clone for feline TNFα which was recently reported (McGraw, 1990). The two feline TNFα clones differ by 8 nucleotide base pair (bp) changes which result in 5 amino acid differences in the predicted protein sequence. A search of GenBank and EMBL determined that the feline TNFα cDNA consensus sequence had a 90, 86, 85, 82 and 83 percent overall match with human, porcine, ovine, mouse and goat TNFα cDNAs, respectively. The protein‐coding sequence for feline TNFα from start to stop codon is 702 bp in length and encodes a predicted protein of 233 amino acids with a molecular weight of approximately 25,446 daltons (precursor form of secreted form of TNFα).

The protein‐coding sequence for feline IL‐1β is 804 bp long and encodes a predicted protein of 267 amino acids with a molecular weight of 31,892 daltons (precursor form of secreted IL‐1β). The feline IL‐1β cDNA consensus sequence had an overall match of 79, 76, 77.5 and 77 percent with IL‐1β cDNA from human, bovine, rabbit and murine species, respectively.     

Purification of follicular regulatory protein: possible plasminogen identity

A partially purified protein (the SR fraction) of porcine and human origin has been extensively characterized as Follicular Regulatory Protein (FRP). In the current study, 1A8D5, one of several monoclonal antibodies raised against FRP, was used to further purify the protein. The monoclonal antibody cross-reacted only with porcine plasminogen, a key fibrinolytic proenzyme. A commercial polyclonal antibody for human plasminogen confirmed the relationship between plasminogen and bands of the SR fraction of the porcine follicular fluid. Sequencing of the N-terminal amino acids (54 kd) of the SR fraction indicated that it shared 100% identity with the short form of porcine plasminogen chain A and 93% identity to human plasminogen. Moreover, we demonstrated that this purified protein from human follicular fluid inhibited aromatase activity of granulosa cells, a key biological property of FRP. Given that plasminogen possesses most of the proposed properties of the protein termed FRP, we conclude that FRP is likely plasminogen itself or a plasminogen-related protein and not a novel protein.     

Molecular cloning,expression pattern and chromosomal mapping of pig CD69

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

Cloning and characterization of Disc1, the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1)

We cloned the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1), a candidate gene for schizophrenia. Disc1 is 3163 nucleotides long and has 60% identity with the human DISC1. Disc1 encodes 851 amino acids and has 56% identity with the human protein. Disc1 maps to the DISC1 syntenic region in the mouse, and genomic structure is conserved. A Disc1 splice variant deletes a portion of Disc1 beginning at amino acids orthologous to the human truncation. Bioinformatic analysis and cross-species comparisons revealed sequence conservation distributed across the genes and conservation of leucine zipper and coiled-coil domains in both orthologs. In situ hybridization in adult mouse brain revealed a restricted expression pattern, with highest levels in the dentate gyrus of the hippocampus and lower expression in CA1-CA3 of the hippocampus, cerebellum, cerebral cortex, and olfactory bulbs. Identification of Disc1 will facilitate the study of DISC1's function and creation of mouse models of DISC1 disruption.