Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis).

Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl alpha-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.

Concanavalin-A-extractable non-mucous glycoprotein concentrations in gallbladder bile of cholesterol gallstone patients.

BACKGROUND: The relationship between protein concentrations and the nucleation activity of bile in cholesterol gallstone patients has already been investigated. Nucleation promoters are mucins and concanavalin A (Con-A)-extractable glycoproteins. Nucleation inhibitors are apolipoproteins. We wanted to investigate whether a change in concentration of apolipoprotein A-I (Apo A-I) or Con-A in the bile of cholesterol stone carriers is dependent on the nucleation time. METHODS: Total protein was measured by fluorescence photometry, and Con-A-extractable glycoproteins were separated by their affinity to lectins and measured by photometry. Apolipoproteins were measured by radioactive competitive protein binding assay. RESULTS: The protein concentrations in our bile samples were 2.41 +/- 1.08 mg/ml for the whole group, 2.73 +/- 1.07 mg/ml for a nucleation time less than 3 days, and 2.04 +/- 1.00 for a longer nucleation time. The concentration of the Con-A fraction accounted for 0.289 +/- 0.096 mg/ml, 0.306 +/- 0.081 mg/ml, and 0.274 +/- 0.109, respectively. The Apo A-I concentration was 52 +/- 64 micrograms/ml; 50 +/- 56 micrograms/ml for a nucleation time less than 3 days and 85 +/- 133 micrograms/ml for a longer nucleation time. CONCLUSIONS: Obviously, individual protein fractions have an effect on the nucleation behaviour of gallbladder bile in cholesterol gallstone patients.

Protein splicing: excision of intervening sequences at the protein level.

Protein splicing is an extraordinary post-translational reaction that removes an intact central "spacer" domain (Sp) from precursor proteins (N-Sp-C) while splicing together the N- and C-domains of the precursor, via a peptide bond, to produce a new protein (N-C). All of the available data on protein splicing fit a model in which these intervening sequences excise at the protein level via a self-splicing mechanism. Several proteins have recently been discovered that undergo protein splicing, and in two such cases, the excised spacer protein is an endonuclease. Such endonucleases are capable of conferring genetic mobility upon the intervening sequences that encodes them. These intervening sequences define a new family of mobile genetic elements that are translated yet remain phenotypically silent by excising at the protein rather than the RNA level.

Mechanism of cleavage at Asn 148 during the maturation of jack bean concanavalin A.

The maturation of the lectin concanavalin A from the jack bean, Canavalia ensiformis, involves an unusual post-translational cleavage at three internal asparagine residues and the subsequent rearrangement and ligation of two of the resulting fragments. It is presently unclear whether these reactions are enzymatically catalyzed or occur autocatalytically. A specific model for non-enzymatic cleavage has been proposed where the attack of the side chain amide nitrogen atom of asparagine on its alpha-carbonyl carbon cleaves the peptide-bond and leaves a C-terminal succinimide residue. Spontaneous hydrolysis of the succinimide would result in the formation of both terminal asparagine and isoasparagine (aspartic acid alpha-amide) residues. We tested this model by chemically analyzing the C-terminal tryptic peptide of mature concanavalin A that contains the cleavage site residue Asn-148 as its C-terminal residue. We found that only free asparagine was released from this peptide with either elastase or leucine aminopeptidase digestion under conditions that released isoasparagine from a similar synthetic peptide. These results suggest that precursor processing in concanavalin A does not in fact follow a succinimide pathway, although other types of autocatalytic mechanisms remain possible.

The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis.

The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.

Posttranslational processing of concanavalin A precursors in jackbean cotyledons.

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation.

T cell-derived glucosteroid response-modifying factor (GRMFT): a unique lymphokine made by normal T lymphocytes and a T cell hybridoma.

Mouse spleen cells and a murine T cell hybridoma, FS6 14.13.1, produce a glucosteroid response-modifying factor (GRMFT) after stimulation with concanavalin A. GRMFT blocks glucosteroid suppression of helper T cell function and the growth of granulocyte/macrophage progenitor cells in vitro. IL 1 also protects helper T cells and myeloid precursors from glucosteroid suppression. This suggests that GRMFT and IL 1 act congruently to ensure that an effective immune response is generated when endogenous glucosteroid levels are elevated. To understand the role of GRMFT in normal immune responses and in disease states characterized by imbalances in the immune system, we began to purify and characterize GRMFT. GRMFT appears to be distinct from other well-characterized T cell-derived factors. GRMFT is larger than IL 2 as determined by gel exclusion chromatography and is completely separated from IL 2 by isoelectric focusing. Furthermore, purified IL 2 does not have GRMFT activity. Purified IL 3 also lacks GRMFT activity, and conditions that inactivate immune interferon have no effect on GRMFT. Thus, GRMFT is different from IL 2, IL 3, and immune interferon. GRMFT also lacks activity in the thymocyte co-mitogenic assay and is therefore different from IL 1. Finally, FS6 14.13.1 reportedly does not produce TRF or CSF, which suggests that GRMFT is different from these molecules as well.

Soluble factor(s) released by Concanavalin A activated lymph node lymphocytes induce proliferation and maturation of chronic lymphocytic leukaemia (CLL) B lymphocytes.

An active supernatant (Ly Con A) was prepared by stimulating the normal human lymph node lymphocytes with Concanavalin A (Con A). Peripheral blood lymphocytes (PBL) from 3 healthy subjects and from 8 patients with chronic lymphocytic leukaemia (CLL) were cultured in the presence of Con A and Ly Con A. The latter induced a significant DNA synthesis both in normal and CLL lymphocytes. A proliferative response was still present in CLL after T lymphocytes depletion. The Con A and Ly Con A treatment also induced morphological changes in CLL lymphocytes consistent with plasma cell differentiation. In 3 cases the appearance of cytoplasmic light chains was detected by immunofluorescence. These findings suggest that peripheral blood B lymphocytes from CLL patients can be stimulated to maturation when helper factor(s) released by mitogen activated lymph node lymphocytes are provided.

Self recognition of accessory cell Ia determinants is required for the in vitro generation of hapten-specific cytotoxic T lymphocyte responses.

The present study has addressed the involvement of Ia determinants in the in vitro generation of antigen-specific cytotoxic T lymphocyte (CTL) responses. It demonstrated that the in vitro generation of TNP-specific CTL responses strictly requires responder T cell recognition of self-Ia determinants expressed by accessory cells, and that this recognition could be specifically inhibited by monoclonal anti-Ia-antibodies. The generation of TNP-specific CTL responses was unaffected by the presence of anti-I-A antibodies or the absence of accessory cells when cultures were performed in the presence of an exogenous source of T helper cell factors, Con A SN. Thus, these results indicate that T helper cell recognition of self-Ia determinants expressed by accessory cells is required for the generation of TNP-specific CTL responses. These results preclude the possibility that accessory cells perform only immunologically nonspecific roles in the generation of hapten-specific CTL, but instead demonstrate that accessory cells function in such responses as Ia-bearing antigen-presenting cells for the activation of self-Ia-specific T cells.

Augmentation of B-cell responsiveness by a tumor-activated T-cell factor.

The growth of the P815 mastocytoma in syngeneic DBA/2 mice led to an activation of Ly1+2- T cells. These T cells produced a soluble factor or factors in culture which, when added to normal spleen cells or B cells in the presence of syngeneic Ly1 cells, caused a genetically unrestricted augmentation of the plaque-forming response toward sheep red blood cells (SRBC). The culture supernatant of the activated T cells did not support the proliferation of an interleukin-2 (IL-2)-dependent cell, nor exhibit properties of late-acting TRF. Active supernatants appeared to affect directly B cells during the first 48 hr of culture with SRBC in such a way as to make them more responsive to antigen-specific Ly1-cell help.

Studies on B-cell activation in vitro.

When antigen activates B cells with the help of T cells, factors are produced by T cells which induce proliferation and maturation to Ig-secreting cells. T-cell lines and T-cell hybridomas have been obtained which, upon stimulation by antigen or by concanavalin A, produce these B-cell replication and maturation factors. However, their ability to produce these but not other factors, such as the T-cell growth factor, appears to be unstable even upon repeated recloning of the T hybridoma cells. Mitogens are known to replace some of the signals required in T-cell-dependent, antigen-specific activation of B cells. Depletion of cells, however, abolishes the mitogen responsiveness of the B-cell population from spleen. This responsiveness can be repaired when accessory cells such as peritoneal cells, irradiated spleen cells, cells of the macrophage line P388D1 or those from macrophage colonies grown from bone marrow cells with colony-stimulating factor are added back. Soluble factors obtained from different activated macrophages as well as from activated T cells also restore responsiveness. These results argue against a single, non-specific signal model of mitogenic activation of B cells and indicate that this activation is T-cell- but not macrophage-independent.

Positively selected Lyt-2+ and Lyt-2- mouse T lymphocytes are comparable, after Con A stimulation, in release of IL 2 and of lymphokines acting on B cells, macrophages, and mast cells, but differ in interferon production.

Purified mouse T lymphocytes were separated into Lyt-2+ and Lyt-2- populations by the procedure of panning, in which a monoclonal rat anti-Lyt-2 antibody and dishes coated with affinity-purified mouse anti-rat Ig antibodies were used. The populations obtained were 95 to 99% pure as determined by immunofluorescence. Graded doses of these T cells were cultured with optimal mitogenic doses of concanavalin A and the 0 to 24 and 24 to 48-hr culture supernatants were collected. The dose-curve assays of the supernatants of Lyt-2+ and Lyt-2- cells showed comparable activity in interleukin 2 (IL 2) and T cell-replacing factor (TRF), assayed on antigen-stimulated culture of T-depleted spleen cells. Limiting dilution assays of IL 2-secreting precursor cells stimulated by Con A showed a high frequency of precursors in both populations, slightly higher among Lyt-2- cells. The supernatants also contained comparable levels of IPA (inducer of plasminogen activator production by the macrophages), MAF (macrophage-activating factor, assayed by induction of their cytolytic function), and MCGF (mast cells growth factor, assayed on a mast cell line). IPA and MAF were not produced with the same kinetics and in the same T cell concentration conditions as IL 2 and TRF. In contrast, interferon was principally produced by the Lyt-2+ cells.

Inhibition of the production of a soluble helper mediator by cyclosporin A results in the failure to generate alloreactive cytolytic cells in mixed-lymphocyte culture.

The mechanism of action of cyclosporin A (CsA) in inhibiting the induction of alloreactive cytolytic T lymphocytes (CTL) in mixed-lymphocyte culture (MLC) was investigated. CsA at concentrations of 10(-3) to 10(-1) micrograms/ml completely prevented the generation of CTL. However, the addition of culture supernatants from mitogen-activated lymphocytes to MLC not only significantly reversed the suppressive effect of CsA but also fully restored the reactivity of lymphocytes already treated with CsA. By measuring the presence of a soluble helper mediator (SHF) in MLC supernatants, we found that CsA-treated lymphocytes produced no SHF, possibly interleukin 2 (IL-2). The effect of CsA on receptors for IL-2 was subsequently studied and it was found that the binding capacity of 125I-labeled IL-2 to lymphocytes was not altered by the presence of CsA. These findings suggest that the prevention of helper cells from producing SHF, rather than the inhibition of the response of effector cells to SHF, is a possible explanation for the immunosuppression mediated by CsA.

Analysis of the response of B cells from CBA/N-defective mice to nonspecific T cell help.

We have investigated the induction of antibody responses to erythrocyte (RBC)-bound antigens in the (CBA/N x B10)F1 mouse. Male B cells, which express the CBA/N defect, were shown to be unresponsive to RBC antigens when the delivered T cell helper activity was solely nonspecific. Thus we demonstrated that defective B cells did not respond to concanavalin A supernatants or bystander helper activity, in spite of the fact that CBA/N-defective mice could produce these T cell activities. The defective B cell did not respond to RBC-bound antigen in the presence of RBC-primed T cells, although the magnitude of this response was usually twofold less than normal controls. The insensitivity of CBA/N defective B cells to nonspecific T cell helper activities seemed to involve at least the inability of RBC antigens to activate defective B cells in the absence of antigen-specific T cell help.

Helper T-cell-replacing factors and T-cell-growth factors produced by concanavalin A-stimulated and pokeweed mitogen-stimulated mouse spleen cells.

Variables affecting helper factor induction by two T-cell mitogens, concanavalin A (Con A) and pokeweed mitogen (PWM), were examined. It was found that both factors are capable of reconstituting T-cell-dependent antibody response as well as enhancing mitogen-stimulated thymocyte proliferation. Detectable factor production is obtained within 6-12 h of culture; Con A factor production reaches a peak usually around 24 h, while no factor activity is detectable after 72 h of culture period. PWM factor production persists throughout 72 h of culture. Con A factor production can be rescued at culture periods beyond 24 or 48 h by using supraoptimal Con A doses. Con A factor, if incubated at 37 degrees C under cell-free conditions in a serum-free medium, shows a rapid rate of decay; in the presence of cells the decay is considerably reduced.

Products of activated lymphocytes. I. The use of radiolabeling techniques in the characterization and partial purification of the migration inhibition factor of the guinea pig.

General methods were developed and applied to the biosynthesis and purification of products of activated lymphocytes available in minute quantities. The activity studied here was the migration inhibitory factor (MIF) produced by purified protein derivative (PPD)- or concanavalin A (Con A)-stimulated lymphocytes obtained from one guinea pig or less. The methods selected yielded results in terms of two chemical parameters characteristic of the molecules involved, namely K(d) on Sephadex G-75 and isoionic point, pI, on isoelectric focusing. When supernatants were fractionated on G-75 columns, there were several areas even in control supernatants which produced migration inhibition relative to medium controls. However, in PPD- and Con A-stimulated supernatants, at least one peak of MIF activity was found solely in the stimulated cultures, with a K(d) of 0.15. A double-labeling technique was used to characterize the proteins of this peak. Control, unstimulated cultures were labeled with [(14)C]leucine and stimulated cultures were labeled with [(3)H]leucine. After mixing the supernatants and G-75 filtration, a major "ratiolabeled" broad peak. i.e. one with increased (3)H/(14)C ratio, was found. When a narrow portion of this peak about K(d) 0.15, containing most of the MIF activity, was subjected to analytical isoelectric focusing, all of the label was associated with proteins of lower net charge than albumin. A unique ratiolabeled peak was found in PPD- and Con A-stimulated fractions with a pI of approx. 5.3. A micropreparative isoelectric focusing technique was developed and yielded MIF activity in the same region as the major ratiolabeled peak. Further study will be required to ascertain whether the ratiolabeled protein is MIF. By following the K(d), pI, and (3)H/(14)C labeling ratio, at least 14 products of activated lymphocytes, synthesized either de novo or in increased amounts, could be distinguished.