Molecular cloning of IgE-binding fragments of Alternaria alternata allergens.

Exposure to the hyphomycete Alternaria alternata is recognized as an important risk factor for asthma. IgE immunoblotting has been used to catalogue the number and Mr of allergens in A. alternata extracts, with estimates ranging from 10 to 30, although few are present in nearly all extracts studied. Several A. alternata allergens have been cloned, including a subunit of the major allergen Alt a 1, ribosomal P2 phosphoprotein, aldehyde dehydrogenase and a yeast YCP4 homolog. We have cloned two sequences encoding IgE-binding fragments of allergens from an A. alternata lambda gt11 cDNA library using pooled atopic sera from A. alternata-sensitive individuals. One is homologous to a region near the C-terminus of hsp70 from Cladosporium herbarum; a near-complete isoallergen variant of A. alternata ribosomal P2 protein was also cloned. Their lacZ fusion proteins had reactivities of 5 and 14%, respectively, with individual atopic sera, indicating that the corresponding allergens are both minor. This study describes one new A. alternata allergen candidate and implicates ribosomal P2 protein as an allergen thtat is stable between independently isolated clones.

Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit.

Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of >90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.

An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum.

BACKGROUND: Extracts of Cladosporium herbarum, a major source of fungal aeroallergens, exhibit a complex profile of IgE-binding proteins. Yields of conventionally purified allergens from this mold have been insufficient to permit further molecular analyses. OBJECTIVE: To enhance and simplify the purification of allergens from C. herbarum, we have sought to use recombinant DNA techniques to clone, identify and bacterially express immunoselected C. herbarum allergens. METHODS: We constructed a cDNA library in lambda ZAP II using mRNA isolated from C. herbarum. From this library, phage clones encoding a new allergen were immunoselected using pooled human atopic IgE. The cloned cDNA was excised from the phage vector as a recombinant pBluescript II SK-phagemid and sequenced. Expression of the recombinant allergen was carried out in E. coli XL1-blue transformants of the phagemid. Bacterial lysates from cells induced to express the cloned allergen were immunoblotted and probed with individual human atopic IgEs. RESULTS: The cDNA clone encodes a 278 amino acid polypeptide homologous to the C-terminal portion of 70 kDa heat shock protein (hsp 70). The polypeptide possesses features common to other hsps 70, i.e. a similar hydropathic profile and a variable C-terminal region with conserved sequence at the very C-terminus. Binding of the recombinant peptide to IgE from 38% of atopic sera or plasma from individuals allergic to C. herbarum was demonstrated. CONCLUSION: These results indicate that amino acid substitutions are relatively conserved even in the variable C-terminal regions of hsp 70 species. Thus, this study should draw attention to the possibility of induction of anaphylactic responses in a sensitized individual when hsp 70 from any pathogenic species is administered for vaccination.

N-terminus of a major allergen, Alt a I, of Alternaria alternata defined to be an epitope.

A 20mer peptide representing the N-terminus of a major allergen, Alt a I, of Alternaria alternata was synthesized and examined for its antibody binding and antibody induction activities. Alt a I peptide-BSA conjugate reacted with both human IgE and rabbit anti-Alt a I IgG in ELISA, albeit the binding of the peptide to IgE was relatively weak. Control conjugate showed no antibody binding. These results indicated that the N-terminus of Alt a I is an antibody binding site. Moreover, peptide-KLH conjugate and nonconjugated peptide induced both IgG and IgE antibodies in Balb/c mice that recognized both native Alt a I allergen and peptide-BSA conjugate. Since the free peptide was able to induce antibodies in vivo, the peptide may also possess a T cell epitope.

Immunoselection of GRP94/endoplasmin from a KNRK cell-specific lambda gt11 library using antibodies directed against a putative heparanase amino-terminal peptide.

Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix-degrading enzymes normally involved in embryonic morphogenesis, tissue remodelling, angiogenesis and wound healing. Such enzymes include endoglycosidases that degrade heparan sulfate (HS) in endothelial basement membrane, as well as better characterized proteases. Heparanase, an endo-beta-D-glucuronidase initially detected in B16 melanoma cells, has been described as a M(r) 96,000 glycoprotein with pI of 5.2, and has been immunolocalized to the cell surface and cytoplasm. We have utilized a polyacrylamide-gel-based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v-K-ras, exhibits HS-degrading activity similar to that of B16F10 mouse melanoma cells. To immunoselect heparanase-expressing clones from a KNRK-cell-specific lambda gt11 cDNA library, we have also prepared a rabbit anti-serum directed against a putative amino-terminal peptide of B16F10 cellular heparanase. Lysogens from one clone expressed a beta-galactosidase fusion protein whose staining with peptide anti-serum was inhibited by competition with excess peptide. Dideoxy-mediated sequencing of the insert termini of this recombinant revealed that it represents a rat homologue of M(r) 94,000 glucose-regulated protein (GRP94/endoplasmin), a molecular chaperone that contains the exact amino-terminal sequence previously attributed to heparanase. Our results call into question the specificity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti-sera.

Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines.

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.

Transformation of normal rat kidney cells by v-K-ras enhances expression of transin 2 and an S-100-related calcium-binding protein.

v-K-ras transformants of normal rat kidney cells (KNRK) exhibit cell surface-related, transformation-specific properties, including cell-surface fibronectin depletion, induction of anchorage- and density-independent growth, and increased synthesis of transforming growth factors alpha and beta. To search for potential distal effectors of v-K-ras-mediated transformation, we prepared a rabbit antiserum directed against intact KNRK cells to immunoprecipitate and compare proteins from detergent lysates and conditioned media of labeled NRK, KNRK, B77-NRK (a v-src transformant) and ts-371-NRK cells (a Ki-MSV encoding a temperature-sensitive p21v-K-ras). Proteins with enhanced expression in both wild-type v-K-ras and v-src transformants included a cell-surface phosphoglycoprotein with apparent Mr of 79,000 (79K) modified from an 85K protein observed in NRK cell lysates, a cytoplasmic 47K and a 10K protein, and a 57K secreted glycoprotein. A KNRK-specific 21K membrane-associated protein and secreted 59K and 36K secreted glycoproteins were also detected. The expression of the 36K and 59K proteins best correlated with temperature-dependent activation of the ts-371-NRK p21v-K-ras. Immunoselection of recombinant clones from a KNRK-specific lambda gt11 cDNA library allowed identification of the 59K and 10K proteins as transin 2 and an S-100-related calcium-binding protein identified as p9Ka/42A but not previously associated with oncogenic transformation of rat cells. Transin 2 detection by a cell-derived antiserum may also suggest the presence of specific cell-surface binding sites for this enzyme.

Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras.

We studied the effects of simultaneous treatment with 0.1 mM N6, O2'-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.