Cloning and structural analysis of two highly divergent IgA isotypes, IgA1 and IgA2 from the duck billed platypus, Ornithorhynchus anatinus.

To trace the emergence of modern IgA isotypes during vertebrate evolution we have studied the immunoglobulin repertoire of a model monotreme, the platypus. Two highly divergent IgA-like isotypes (IgA1 and IgA2) were identified and their primary structures were determined from full-length cDNAs. A comparative analysis of the amino acid sequences for IgA from various animal species showed that the two platypus IgA isotypes form a branch clearly separated from their eutherian (placental) counterparts. However, they still conform to the general structure of eutherian IgA, with a hinge region and three constant domains. This indicates that the deletion of the second domain and the formation of a hinge region in IgA did occur very early during mammalian evolution, more than 166 million years ago. The two IgA isotypes in platypus differ in primary structure and appear to have arisen from a very early gene duplication, possibly preceding the metatherian eutherian split. Interestingly, one of these isotypes, IgA1, appears to be expressed in only the platypus, but is present in the echidna based on Southern blot analysis. The platypus may require a more effective mucosal immunity, with two highly divergent IgA forms, than the terrestrial echidna, due to its lifestyle, where it is exposed to pathogens both on land and in the water.

Isolation of transcriptionally active umbilical cord blood-derived basophils expressing Fc epsilon RI, HLA-DR and CD203c.

BACKGROUND: Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils. METHODS: To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining. RESULTS: The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcepsilonRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24+/-5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95+/-0.5%) and recovery (59+/-1.5%) of cultured basophils. CONCLUSIONS: We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

Mice lacking histidine decarboxylase exhibit abnormal mast cells.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Characterization of the gene encoding mouse mast cell protease 8 (mMCP-8), and a comparative analysis of hematopoietic serine protease genes.

Serine proteases are important granule constituents in several of the major hematopoietic cell lineages. We present here the nucleotide sequence of the gene encoding mouse mast cell protease 8 (mMCP-8). mMCP-8 was initially isolated as a cDNA from a mouse mast cell line, but has recently been found to be expressed primarily by mouse basophils. mMCP-8 and its rat homologues, rMCP-8, -9, and -10, form a new group of mast cell/basophil proteases, which are more closely related to the T-cell granzymes and neutrophil cathepsin G than to the mast cell tryptases and chymases. A dot matrix comparison of the mMCP-8 gene with other closely related hematopoietic serine protease genes shows detectable homology only in the exonic regions of the genes. No indication for conservation in the promoter region or introns was observed. This latter finding indicates that the upstream regulatory region has evolved at a relatively high rate. However, despite the low degree of direct sequence conservation, no major differences in the sizes of introns or exons were observed between mMCP-8 and genes for the closest related hematopoietic serine proteases, the mouse T-cell granzymes and cathepsin G, indicating that after evolutionary separation from the T-cell granzymes and cathepsin G, the majority of mutations primarily involved single base pair substitutions or short insertions or deletions.

Molecular cloning and preliminary functional analysis of two novel human KRAB zinc finger proteins, HKr18 and HKr19.

cDNA clones encoding two novel human KRAB zinc finger proteins, HKr18 and HKr19, were isolated from a human testis cDNA library. Their corresponding genes were later identified in sequences originating from chromosomes 19 and 7, respectively. On the basis of the collected information from gene and cDNA sequences, Hkr18 was found to be a protein of 94 kDa with 20 zinc finger motifs in its C terminus. The HKr19 is a smaller protein, with a molecular weight of 56 kDa containing 11 zinc finger motifs. Both HKr18 and HKr19 contained a KRAB A as well as a KRAB B domain in their N termini. Northern blot analysis showed expression of HKr18 in all human tissues tested, indicating a ubiquitous expression pattern. In contrast, HKr19 showed a more restricted tissue distribution, with detectable expression primarily in testis and fetal tissues. The HKr19 protein is a member of the large ZNF91 subfamily of KRAB zinc finger genes. A PCR-based analysis of the expression of HKr19 and other closely related genes showed that lymphoid, myeloid, and nonhematopoietic cells expressed different sets of these genes. This latter finding indicates that some members of the ZNF91 family may be involved in regulating lineage commitment during hematopoietic development. Transfection of various parts of HKr19 into human embryonic kidney cells (HEK 293 cells) showed that the entire protein and its zinc finger region were toxic to these cells when expressed at high levels. In contrast, the KRAB domain and the linker region seemed to be well tolerated.

Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 beta.

The Krüppel-associated box (KRAB) domain, originally identified as a 75-aa sequence present in numerous Krüppel-type zinc-finger proteins, is a potent DNA-binding-dependent transcriptional repression domain that is believed to function through interaction with the transcriptional intermediary factor 1 (TIF1) beta. On the basis of sequence comparison and phylogenetic analysis, we have recently defined three distinct subfamilies of KRAB domains. In the present study, individual members of each subfamily were tested for transcriptional repression and interaction with TIF1 beta and two other closely related family members (TIF1 alpha and TIF1 gamma). All KRAB variants were shown, (i) to repress transcription when targeted to DNA through fusion to a heterologous DNA-binding domain in mammalian cells, and (ii) to interact specifically with TIF1 beta, but not with TIF1 alpha or TIF1 gamma. Taken together, these results implicate TIF1 beta as a common transcriptional corepressor for the three distinct subfamilies of KRAB zinc-finger proteins and suggest a high degree of conservation in the molecular mechanism underlying their transcriptional repression activity.

Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus.

To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.

Murine mast cell lines as indicators of early events in mast cell and basophil development.

To study early events in mast cell / basophil development, the phenotype of a panel of murine cell lines at various stages of differentiation was determined. Based on the expression on various mast cell-specific proteases and several additional hematopoietic differentiation markers, the cell lines CFTL-15 and MCP5 / L were clearly identified as mast cells, although with a relatively immature phenotype. These two cell lines express the high-affinity IgE receptor alpha-chain, the mouse mast cell protease (MMCP)-5 and the carboxypeptidase A (CPA). Bone marrow-derived mast cells and the transplantable mast cell tumor MTC were shown to express the IgE receptor alpha-chain, MMCP-5 and CPA, as well as the mast cell tryptase MMCP-6 and the chymase MMCP-4, a protease expressed only during late stages of mast cell differentiation. These two cell types thus display a more mature mast cell phenotype. In contrast, the cell lines P815 and 32D cl3 did not express any mast cell differentiation markers. Interestingly, the IC-2 cell line was shown to express several markers for immature mast cells and in addition MMCP-8, a serine protease which may represent a marker for mouse basophils. By antibody staining, almost all IC-2 cells were shown to express MMCP-8. This indicates that individual cells may simultaneously express both mast cell and basophil markers. Moreover, these findings suggest that an early branch point in hematopoietic development where mast cells and basophils have a common precursor cell may exist.

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.