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J W Goding

Publications and source records attributed to J W Goding.

At least 37 records · Page 2Linked to original sources
PubMed1987

Plasma cell membrane glycoprotein PC-1. Primary structure deduced from cDNA clones.

The PC-1 protein is a membrane glycoprotein that is selectively expressed on the surface of antibody-secreting cells. Previous work has shown that it consists of two apparently identical disulfide-bonded polypeptides, each of molecular weight approximately 120,000. We now describe the sequence of PC-1 mRNA and protein. The PC-1 protein is shown to consist of 905 amino acids and to have an uncommon transmembrane orientation. The NH2-terminal 58 residues are intracellular and the COOH-terminal 826 residues are extracellular. A cysteine-rich region of 85 amino acids lies adjacent to the extracellular surface of the membrane and appears to have arisen by exon duplication. In common with other membrane glycoproteins with this orientation, there is no obvious signal sequence other than the transmembrane segment. The PC-1 protein therefore has an overall structure and membrane orientation that resembles those of the transferrin receptor, the asialoglycoprotein receptor, and the Ia invariant chain.

Amino Acid Sequence↗
PubMed1987

Leishmania major: a very sensitive dot-blot ELISA for detection of parasites in cutaneous lesions.

There is a need for accurate, rapid and early diagnosis of leishmaniasis, which would distinguish between the benign and severe forms of the disease. We have used a monoclonal antibody directed to a polymorphic, species-specific antigen in diagnostic assays for leishmaniasis. The dot-blot enzyme-linked immunosorbent assay described here can detect as few as 300 culture promastigotes and 20,000 amastigotes of Leishmania major with no cross-reaction with other species and no background from skin macrophages or other cells. This level of sensitivity is sufficient to detect parasite antigen aspirated in a few microliters of liquid from small lesions in mice. This assay could form the basis for a sensitive, rapid and inexpensive dip-stick test for large-scale use for the diagnosis and epidemiology of cutaneous leishmaniasis.

Animals↗
PubMed1986

Use of microbore high-performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing. Structural studies on the murine plasma cell antigen PC-1.

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.

Amino Acid Sequence↗
PubMed1985

Heterogeneous glycosylation of murine transferrin receptor subunits.

The N-linked glycosylation of the murine receptor for transferrin has been investigated. Previously we have found that purified receptors appear as two bands after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Coomassie blue staining [van Driel, I.R., Stearne, P.A., Grego, B., Simpson, R.J. and Goding, J.W. (1984) J. Immunol. 133, 3220-3224]. In the current report we show that the two bands are due to different glycosylation of individual receptor molecules. The receptors have three asparagines to which N-linked glycans can be added, but only two sites are glycosylated in all receptors. The level of glycosylation of the third site varies depending on cell line or tissue. Limited endoglycosidase digestion of mature receptors indicates that differential glycosylation probably occurs at only one particular asparagine residue. Possible mechanisms that could result in such a glycosylation pattern are discussed.

Animals↗
PubMed1985

A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor.

We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.

Amino Acid Sequence↗
PubMed1985

The Leishmania receptor for macrophages is a lipid-containing glycoconjugate.

The glycoconjugate of Leishmania major recognized by the monoclonal antibody WIC-79.3 exists in two forms. The cellular form associated with the promastigote is a population of amphipathic molecules consistent with membrane insertion. In contrast, the extracellular form mainly consists of hydrophilic molecules, and probably arises by cleavage of the cellular form by an endogenous phospholipase. The hydrophilic population of extracellular glycoconjugate molecules binds specifically to macrophages but not to T or B lymphoid cells. Binding of the glycoconjugate and also intact promastigotes to macrophages in vitro is specifically inhibited by Fab fragments of WIC-79.3. These data indicate that the L. major glycoconjugate is the parasite receptor for macrophages, and hence the molecule directly involved in the initiation of infection.

Animals↗
PubMed1985

Murine plasma cell membrane antigen PC-1: molecular cloning of cDNA and analysis of expression.

PC-1 is a membrane glycoprotein expressed selectively on murine antibody-secreting plasma cells. Previously, we have obtained partial amino acid sequence data for this protein. Here, we describe the use of these data in the isolation of PC-1 cDNA clones. To avoid the "3'-bias" of conventional cDNA libraries we constructed a cDNA library in lambda gt10 by priming the first strand of cDNA synthesis with random hexadeoxynucleotides. The library was screened with oligonucleotide probes, 17 nucleotides in length, the design of which was based on the amino acid sequence data. Two cDNA clones of 1.0 and 0.9 kilobase pairs (lambda RR3 and lambda RR20, respectively) were isolated. Both contained a sequence encoding the 15-residue tryptic peptide that was used to derive the sequences of the oligonucleotide probes. lambda RR3 cDNA hybridized to an approximately equal to 3.5-kilobase mRNA that was present in plasmacytomas, spleen, and liver but not in other cell types screened. We were unable to detect PC-1 mRNA or protein in mouse brain. In the spleens of mice chronically infected with Mesocestoides corti, PC-1 mRNA was present at 2.5-fold higher levels than found in normal mouse spleens, whereas immunoglobulin mRNA levels were 15-fold higher. Southern blot analyses revealed the presence of only one gene copy per haploid mouse genome. Restriction fragment length polymorphisms were detected in genomic DNA from mice bearing different PC-1 alleles. A related gene is present in rat genomic DNA.

Animals↗
PubMed1985

cDNA cloning of the murine transferrin receptor: sequence of trans-membrane and adjacent regions.

We previously purified the murine transferrin receptor from cultured myeloma cells and determined the amino acid sequence of six tryptic peptides. We now report the cloning of cDNA encoding the murine transferrin receptor. A tryptic peptide containing a region of six consecutive amino acids, all encoded by four or fewer codons, was used to design two overlapping oligonucleotide probes, 17 and 14 nucleotides in length. These probes were used to screen a lambda gt10 cDNA library from NS-1 myeloma cells. Of approximately 400,000 plaques screened, two hybridized strongly to both probes. A subfragment of one clone that hybridized with both oligonucleotide probes was found to encode the tryptic peptide from which the probes were derived, as well as another sequenced tryptic peptide. Comparison of the sequence with that of the human transferrin receptor shows a high degree of conservation of the sequences surrounding and penetrating the membrane, including cysteine residues that may be involved in interchain disulfide bonding and/or covalent attachment of lipid. The current data, when combined with the published sequence of the human receptor, allow assignment of all six tryptic peptides to a single chain, supporting the idea that the receptor is a homodimer. A 4.9-kb messenger RNA was found in several cultured murine and human tumor cell lines, but transferrin receptor messenger RNA was not detectable in murine spleen. An additional RNA species of 2.7 kb was present in approximately equal abundance in the murine myelomas NS-1 and C118 but was absent from T lymphomas TIKAUT, ST-1, and ST-4.

Amino Acid Sequence↗
PubMed1985

Plasma cell antigen PC-1 and the transferrin receptor in mouse, rat, and hamster: serologic and biochemical analysis.

The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.

Animals↗
PubMed1985

The murine plasma cell antigen PC-1: purification and partial amino acid sequence.

The murine plasma cell alloantigen PC-1 is selectively expressed on B lymphocytes in their terminal phase of differentiation into antibody-secreting cells. Previous work on an analytical scale has shown that PC-1 consists of two apparently identical disulfide-bonded polypeptides, each of Mr 115,000. In this paper, we describe the generation of a monoclonal antibody to PC-1 and its use in the preparative isolation of PC-1 by affinity chromatography. Final purification to apparent homogeneity was achieved by preparative polyacrylamide gel electrophoresis. It was estimated that NS-1 myeloma cells possess 1 to 4 X 10(5) PC-1 monomers per cell on their surface. The yield of PC-1 after purification was approximately 10(5) monomers per cell. Purified PC-1 was digested with trypsin, and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. Purified peptides were sequenced with a gas-phase sequencer.

Amino Acid Sequence↗
PubMed1984

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies.

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.

Animals↗
PubMed1984

The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence.

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.

Amino Acid Sequence↗
PubMed1982

Monoclonal antibodies to the murine Ly-2.1 cell surface antigen.

Six monoclonal anti-Ly-2.1 antibodies are described that arose from a single fusion using the spleen from a 129/ReJ mouse immunized with CBA/H thymus cells. The specificity was determined from the strain distribution, and testing of congenic strains where all six monoclonal antibodies detected the Ly-2.1 alloantigen. Furthermore, the antibodies fall into two groups: Group I (four antibodies) detected the expected number of Ly-2+ thymocytes and peripheral T cells, whereas Group II (two antibodies) detected 10% fewer peripheral T cells. Functional studies demonstrated that the Ly-2.1 determinant detected by a Group I antibody (49-31.1) was presented on cytotoxic T cells (Ly-1+2+), on concanavalin A (Con A)-induced suppressor T cells (Ly-1-2+), but absent from helper T cells (Ly-1+2-). One antibody (49-11.1) precipitated a 68,000-75,000 mol.wt glycoprotein, which on reduction yielded 30,000 and 35,000 mol.wt moieties. Thus, by functional, genetic and biochemical criteria these antibodies detected the Ly-2.1 specificity. In addition, a monoclonal, noncytotoxic, IgGl, anti-Thy-1.2 antibody is described.

Animals↗
PubMed1982

Structure of the murine plasma cell alloantigen PC-1: comparison with the receptor for transferrin.

The plasma cell alloantigen PC-1 was isolated from C1.18 myeloma cells by immunoprecipitation and was analyzed by polyacrylamide gel electrophoresis. It was found to consist of two similar or identical disulfide-bonded polypeptide chains, each of Mr 115,000. The mobility of PC-1 in nonequilibrium pH gradient electrophoresis was similar to that of bovine serum albumin (pI 4.9). The PC-1 antigen is therefore similar to the transferrin receptor in Mr, charge, subunit composition, disulfide bonding, and developmental regulation. Similarities can also be detected by peptide mapping with subtilisin, but not with staphylococcal V8 protease. It is suggested that the PC-1 protein and the transferrin receptor may have had a common evolutionary origin, and may have similar functions.

Animals↗