Biomedical subjects

M W Robertson

Publications and source records attributed to M W Robertson.

25 records · Page 2Linked to original sources

PubMed1990

Human IgE-binding protein: a soluble lectin exhibiting a highly conserved interspecies sequence and differential recognition of IgE glycoforms.

IgE-binding protein (epsilon BP) refers to a protein originally identified in rat basophilic leukemia cells by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin equivalent to carbohydrate-binding protein 35 (CBP 35). More recently, its identity to Mac-2, a macrophage cell-surface protein, has been established. cDNA coding for human epsilon BP has been cloned from a human HeLa cell cDNA library and contains an open reading frame of 750 base pairs encoding a 250 amino acid protein. Like the rat and murine counterparts, the human epsilon BP amino acid sequence can be divided into two domains with the amino-terminal domain consisting of a highly conserved repetitive sequence (YPGXXXPGA) and the carboxyl-terminal domain containing sequences shared by other S-type lectins. The human epsilon BP sequence exhibits extensive homology to murine and rat epsilon BP with 84% and 82% identity, respectively. The homology is particularly striking in the carboxyl-terminal domain where 95% identity is found between human and murine sequences in a stretch of over 70 amino acids. A survey of epsilon BP mRNA expression from several lymphocyte cell lines revealed that the level of epsilon BP transcription may reflect a relationship between cell differentiation and epsilon BP expression. Finally, human epsilon BP was purified from several human cell lines and shown to possess lactose-binding characteristics and cross-species reactivity to murine IgE. Surprisingly, three different human myeloma IgE proteins did not show reactivity to human epsilon BP. However, after neuraminidase treatment of each human IgE, pronounced binding to epsilon BP was observed, thereby indicating that only specific IgE glycoforms can be recognized by epsilon BP.

Amino Acid Sequence↗

PubMed1989

Biochemical and immunological comparisons of carbohydrate-binding protein 35 and an IgE-binding protein.

The predicted amino acid sequence of carbohydrate-binding protein 35 (CBP35; Mr approximately 35,000), a galactose-specific lectin in many mouse and human cells, has been compared to the predicted sequence of an IgE-binding protein (epsilon BP) originally identified in rat basophilic leukemia cells. The sequences of the two proteins showed that: (a) 85% of the amino acid residues are identical; (b) the polypeptide chains are comprised of two distinct domains; and (c) highly conserved internal repetitive sequences are present. Consistent with these observations, antisera raised against CBP35 or against a synthetic peptide derived from the epsilon BP sequence cross-reacted with both proteins. Moreover, fractionation of extracts of mouse 3T3 fibroblasts over an IgE-Sepharose affinity column showed that CBP35 bound to IgE; this binding was reversed by addition of lactose. Conversely, fractionation of extracts of rat basophilic leukemia cells over an affinity column of Sepharose derivatized with N-(epsilon-amino-caproyl)-D-galactosamine showed that epsilon BP was a galactose-binding protein. Furthermore, epsilon BP bound to IgE-Sepharose could be eluted by lactose. Finally, transcription and translation in vitro of the cDNA corresponding to epsilon BP yielded a polypeptide containing carbohydrate-binding activity. All of these results strongly support the conclusion that CBP35 and epsilon BP are mouse and rat homologues, respectively.

Animals↗

PubMed1988

IgE-binding protein. Subcellular location and gene expression in many murine tissues and cells.

We show that IgE-binding protein (epsilon BP) is found primarily in the cytoplasm of rat basophilic leukemia (RBL) cells and COS-1 cells transfected with epsilon BP cDNA. Antibodies to a synthetic peptide internal to epsilon BP were generated that specifically recognized epsilon BP by protein immunoblotting. These antibodies also bind the surface of RBL cells. Surprisingly, blot hybridization analysis of RNA from nine various normal rat tissues showed that the epsilon BP gene is transcribed in all the tissues tested as well as in a mouse macrophage-like cell line.

Animals↗

PubMed1988

IgE structure-function relationships defined by sequence directed antibodies induced by synthetic peptides.

Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.

Amino Acid Sequence↗

PubMed1988

cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

Amino Acid Sequence↗

PubMedDate not supplied

Expression and function of an IgE-binding animal lectin (epsilon BP) in mast cells.

epsilon BP (IgE-binding protein) is a 31,000 M(r) protein originally identified in rat basophilic leukemia (RBL) cells. The protein is composed of two domains with the amino-terminal domain containing a highly conserved repetitive sequence and the carboxyl-terminal domain containing consensus sequences shared by other beta-galactoside-binding soluble lectins. The protein has wide tissue distribution, is found on cell surfaces and in extracellular milieu. By combined efforts from several research groups including ours a multifunctional nature of this lectin began to emerge. This review emphasizes the following characteristics of epsilon BP: (i) epsilon BP is secreted by cells such as macrophages; (ii) like many other lectins, epsilon BP functions at least bivalently; (iii) epsilon BP has specificity for distinct oligosaccharide structures that have a terminal galactose not masked by sialic acids; and (iv) in addition to binding IgE, epsilon BP binds to surfaces of various cell types via lectin-carbohydrate interaction. Importantly, epsilon BP binds to the IgE receptor on mast cells. We propose that epsilon BP can function as a modulatory protein on various cells by cross-linking critical cell surface glycoproteins. The proposed action of epsilon BP on mast cells is presented as a model.

Amino Acid Sequence↗

PubMedDate not supplied

Use of a tissue-specific promoter for targeted expression of the herpes simplex virus thymidine kinase gene in cervical carcinoma cells.

Molecular chemotherapy strategies have been developed for a number of epithelial malignancies based on selective delivery and expression of a toxin-encoding gene into the cancer cells. To date, these strategies have not been explored in the context of carcinoma of the cervix, despite the fact that a variety of factors suggest this as an appropriate disease for this gene therapy approach. One limitation in this respect is that appropriate tissue-specific promoters for selective toxin gene expression have not been defined for cervical carcinoma. In this regard, the secretory leukoprotease inhibitor (SLPI) gene has been shown to be constitutively expressed in many epithelial carcinoma cells including the uterine cervix. Thus, we investigated the utility of the SLPI gene as a tissue-specific promoter for regulatory control of the herpes simplex virus thymidine kinase gene for in vitro treatment of cervical carcinoma cells. For this analysis, a gene construct was derived with the herpes simplex virus thymidine kinase gene under regulatory control of the 5' upstream regions of the SLPI gene. Transient transduction of three human cervical carcinoma cell lines with the SLPI-thymidine kinase (TK) construct was followed by treatment with the prodrug ganciclovir. Crystal violet staining was subsequently used to assess cell viability. In this analysis, it was shown that the SLPI-TK construct directed TK-mediated killing in two of three tested cervical cell lines, with the two cell lines being positive for SLPI. In addition, mixing experiments established that cervical carcinoma cells could exhibit a bystander effect which potentially augments the efficacy of molecular chemotherapy approaches. These findings may allow for the development of efficacious, target-specific, toxin gene therapy strategies for cervical carcinoma in human patients.

Carcinoma↗