Search PubMed⌕ Search

Biomedical subjects

H W Davidson

Publications and source records attributed to H W Davidson.

28 records · Page 2Linked to original sources
PubMed1990

The antigen processing pathway in B lymphocytes.

Monovalent antigen bound to membrane immunoglobulin on human B lymphoblastoid cells is endocytosed through coated pits and passes sequentially from a site which actively recycles membrane to the cell surface and then to a proteolytically active processing site. A high affinity of immunoglobulin for antigen precludes antigen dissociation following endocytosis and results in the processing of an antigen/Ig complex yielding an epitope-specific pattern of processing. Processing of the immunoglobulin also occurs and is enhanced by occupancy with monovalent antigen.

Antigens↗
PubMed1989

Epitope-directed processing of specific antigen by B lymphocytes.

Proteolytic processing of specific antigen was studied using Epstein Barr virus transformed B-lymphoblastoid cells expressing membrane IgG against tetanus toxin. As previously reported (Watts, C., and H.W. Davidson. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1937-1945), receptor-mediated endocytosis of monovalent antigen bound at 0 degrees C began immediately upon shifting the cells to 37 degrees C. In contrast, degradation of antigen, assessed either by the release of acid-soluble radiolabel into the incubation medium, or by SDS-PAGE analysis of total cell-associated antigen, proceeded after a lag of 10-20 min. Degradation was abolished by exposure of the cells to metabolic inhibitors, or by incubation at 20 degrees C, and inhibited in a dose-dependent fashion by chloroquine and by the lysosomal protease inhibitors leupeptin, E-64, and pepstatin A. Analysis of the cell-associated radiolabel by SDS-PAGE and autoradiography after incubations at 37 degrees C revealed the time-dependent generation of distinct antigen fragments. Virtually quantitative immunoprecipitation of these fragments was obtained using a monoclonal anti-human IgG antibody, indicating that the antigen/mIg complex is the initial substrate for processing. We show that the pattern of fragmentation observed varies from one B cell line to another (a) depending on the epitope through which the antigen is bound and endocytosed and (b) depending on whether additional epitopes in the antigen are complexed with anti-tetanus Fabs. The implications of these results for the presentation of major histocompatibility complex restricted antigen fragments, and for intracellular trafficking of ligand/receptor complexes are discussed.

Antigen-Presenting Cells↗
PubMed1988

Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases.

Insulin is produced from an inactive precursor, proinsulin, through initial endoproteolytic cleavage at sites marked by pairs of basic amino-acid residues. We report here that lysates of insulin secretory granules contain two distinct Ca-dependent acidic endoproteases; one (type I) cleaving exclusively on the C-terminal side of Arg 31.Arg 32 (B-chain/C-peptide junction), the other (type II) preferentially on the C-terminal side of Lys 64.Arg 65 of proinsulin (C-peptide/A-chain junction). The Ca and pH requirements of these proteinases suggested that the type-II proteinase would be active in the Golgi apparatus and the secretory granule, whereas type-I activity would be compatible only with the intragranular environment. Kinetic analyses of (pro)insulin conversion intermediates in [35S]methionine-pulsed rat islets support this supposition. Our results suggest a simple mechanism whereby different dibasic sites can be cleaved in different cellular compartments. In conjunction with the regulation of the ionic composition of such compartments and the operation of post-Golgi segregation, our results also suggest how proteolytic conversion of diverse proproteins destined for different cellular sites can occur differentially and in a regulated manner.

Animals↗
PubMed1988

Endocytosis and recycling of specific antigen by human B cell lines.

Human B cell lines expressing membrane immunoglobulin specific for tetanus toxoid/toxin were used to study the receptor-mediated endocytosis of antigen. Monovalent antigen, initially bound to cell surface immunoglobulin at 0 degree C, was rapidly endocytosed upon warming the cells to 37 degrees C. The kinetics of endocytosis of antigen were independent of the number of occupied binding sites and indicated a half-life for antigen on the cell surface of 8.5 min. Endocytosis of antigen apparently ceased after approximately 15 min at 37 degrees C, although some 40-50% remained on the cell surface at this time. We show, using biotinylated antigen and an avidin detection assay, that this is due to recycling of antigen to the cell surface. By labelling the antigen on the cell surface with Fabs against different epitopes we show that antigen continues to be endocytosed for at least 1 h after the initial rapid phase of endocytosis, again indicating that there must be recycling of immunoglobulin/antigen complexes. As a consequence of the stable interaction between antigen and membrane immunoglobulin, the capacity of the cells to accumulate antigen was limited when the synthesis of membrane immunoglobulin was blocked; under these conditions only 2-3 times as much antigen was endocytosed and degraded when antigen was supplied continuously over a 4-h period at 37 degrees C as could be bound to the cells at 0 degree C. These results reveal a rapid and efficient pathway for the endocytosis and recycling of monovalent antigen in B cells.

Antigen-Antibody Complex↗
PubMed1987

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.

Calcium↗
PubMed1987

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing.

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.

Adenoma, Islet Cell↗
PubMed1987

Biosynthesis of betagranin in pancreatic beta-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin.

The biosynthesis of insulin and betagranin, a 20-21 kDa co-secreted chromogranin A-related protein, were investigated in isolated insulinoma cells and islets. The insulinoma tissue processed proinsulin to insulin with kinetics similar to those reported in islet tissue. Unlike islets, however, the insulinoma released almost one-quarter of the newly synthesized proinsulin into the medium 10-40 min after its formation. Betagranin was initially immunoprecipitated as a 100 kDa precursor form, which was indistinguishable from chromogranin A in size and immunoreactivity and by peptide mapping. After an initial lag of 10-20 min, the precursor was converted progressively into betagranin, which appeared to be a stable end product. Formation of betagranin and insulin from their respective precursors followed a parallel course and could likewise be inhibited by NH4+, chloroquine and monensin, added either before labelling or at any point of time up to 15 min after labelling. As with proinsulin, approximately one-quarter of the betagranin precursor was released 10-40 min after synthesis. It is concluded that betagranin is produced by limited proteolysis from a chromogranin A precursor in pancreatic beta-cells by a cellular pathway indistinguishable from that of insulin from proinsulin. Chromogranin A is highly conserved in the N-terminal region represented by betagranin, further suggesting that the biological activity of chromogranin A may reside in a derived peptide rather than in the parent molecule.

Animals↗
PubMed1987

Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).

The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.

Adenoma, Islet Cell↗