Biosynthesis and secretion of the rat core-specific lectin. Relationship of post-translational modification and assembly to attainment of carbohydrate binding activity

A soluble lectin, the core-specific lectin (CSL), is synthesized and secreted by rat hepatocytes and the rat hepatoma cell line, H-4-II-E. This lectin binds mannose and N-acetylglucosamine residues in the "core" region of Asn-linked oligosaccharides. Secretion of the CSL was found to occur over an extended period of time, greater than 4 h being required for secretion of 50% of the lectin (Brownell, M. D., Colley, K. J., and Baenziger, J. U. (1984) J. Biol. Chem. 259, 3925-3932). We have determined that following synthesis in the endoplasmic reticulum, the CSL is rapidly transported to the Golgi where it is retained for an extended period of time prior to secretion. The lectin undergoes two post-translational modifications within the Golgi: an increase from Mr 24,000 to 25,000 and a progressive decrease in pI with an accompanying increase in Mr to a final value of 26,000. The lectin is also assembled into high molecular weight complexes of 150-260 X 10(3) and acquires the ability to bind carbohydrate in the Golgi. In hepatoma cells, the 24,000-25,000 modification is completed 20 min after initiation of synthesis. Assembly of the CSL subunits into high molecular weight complexes, acquisition of carbohydrate binding activity, and the 25,000-26,000 modification occur between 20 and 80 min after initiation of synthesis. These events have slower kinetics in primary hepatocytes and this allowed us to determine that the sequence of these biosynthetic events is: the 24,000-25,000 modification, complex assembly, the 25,000-26,000 modification, and acquisition of carbohydrate binding activity. The 24,000-25,000 modification occurs prior to complex assembly. Complex assembly may occur prior to, or concomitant with, the 25,000-26,000 modification. Assembly into the oligomeric form and the 25,000-26,000 modification correlate with the attainment of carbohydrate binding activity. The kinetics of CSL modification and assembly cannot account for its retention within the Golgi. Interaction with Golgi components either through carbohydrate binding or another interaction, may act to selectively retain the lectin within the Golgi.     

Post-translational modifications of the core-specific lectin. Relationship to assembly, ligand binding, and secretion

The rat core-specific lectin (CSL) or mannan-binding protein is synthesized and secreted by rat hepatocytes and H-4-II-E hepatoma cells. Prior to secretion proline and lysine residues with collagen-like sequences undergo hydroxylation and subsequent glycosylation of hydroxylysine to produce glucosylgalactosylhydroxylysine. Hydroxylation and subsequent glycosylation are inhibited by alpha,alpha'-dipyridyl (Colley, K. J., and Baenziger, U. U. (1987) J. Biol. Chem. 262, 10290-10295). We have used alpha,alpha'-dipyridyl to investigate the role of hydroxylation and glycosylation on interchain disulfide bond formation, assembly of subunits into high molecular weight complexes, attainment of carbohydrate and lipid binding ability, and secretion. Formation of disulfide-bonded dimers and trimers in the endoplasmic reticulum, assembly into high molecular weight complexes in the Golgi, and attainment of carbohydrate binding activity occur in either the presence or absence of these post-translational modifications. The mature fully processed form of the CSL binds hydrophobic matrices and is secreted at a slow, but linear, rate. Inhibition of proline and lysine hydroxylation and hydroxylysine glycosylation prevents CSL secretion and attainment of binding activity for hydrophobic matrices. Secretion of the lectin, although slow, appears to be an active process and may be related to the capacity to interact with membranes and/or lipids. Other proteins known to contain collagen-like sequences such as acetylcholinesterase, pulmonary surfactant apoproteins, and C1q also interact with lipids and/or membranes. The collagen-like domains of these proteins may also play a role in promoting such interactions.     

Identification of the post-translational modifications of the core-specific lectin. The core-specific lectin contains hydroxyproline, hydroxylysine, and glucosylgalactosylhydroxylysine residues

The core-specific lectin (CSL) synthesized and secreted by rat hepatocytes and the rat hepatoma H-4-II-E shows affinity for mannose and N-acetylglucosamine residues in the "core" region of asparagine-linked oligosaccharides. The CSL undergoes two stages of post-translational modification which result in an increase in its Mr from 24,000 to 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have determined that the lectin undergoes hydroxylation of proline and lysine and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GlcGalHyLys). CSL metabolically labeled with [3H]lysine and [3H]proline contains hydroxylated forms of proline and lysine. The mature form of the lectin can also be metabolically labeled with [3H]galactose. alpha,alpha'-Dipyridyl, an inhibitor of collagen prolyl and lysyl hydroxylases, prevents the metabolic incorporation of [3H]galactose and the post-translational increases in the Mr of the CSL, indicating that both events are dependent upon hydroxylation of proline and lysine. Virtually all of the hydroxylysine present in the CSL is recovered as glucosylgalactosylhydroxylysine after alkaline hydrolysis. The post-translational modifications of the CSL place it in a select family of secreted proteins which contain collagen-like sequences, including the pulmonary surfactant proteins, complement component C1q, and the 18 S asymmetric form of acetylcholinesterase.     

Blood pressure in a national birth cohort at the age of 36 related to social and familial factors,smoking, and body mass.

Blood pressure was measured in a birth cohort of 5362 subjects at the age of 36. The prevalence of hypertension in men (blood pressure greater than 140/90 mm Hg) was almost twice that in women, although women received treatment more often. Deaths of fathers of subjects from hypertensive and ischaemic heart disease were associated with significantly higher mean systolic and diastolic pressures in both sexes. Cigarette smoking was not strongly associated with blood pressure in men and not associated at all in women. Of the social factors, low social class of family of origin was associated with high blood pressure in both sexes; but the strongest association was with current body mass, and birth weight also contributed. Differences in blood pressures between the sexes may have been related to protective biological factors, such as endogenous sex hormones, in women and also to differences in types of employment, smoking habits, and body mass. Differences in blood pressures related to the social class of family of origin may reflect long term influences of class differences on diet, exercise, and educational achievement. The importance of measuring secular trends in obesity and blood pressures is emphasised.     

Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for an anti-idiotypic, I-J-positive, I-J-restricted, soluble T suppressor factor

Modulation of pathogenic egg-induced hepatic granuloma formation in chronically Schistosoma mansoni-infected mice is an immunoregulatory process. Adoptive transfer and in vitro studies have demonstrated that this suppression involves various T lymphocyte circuitries, and the participation of soluble suppressor factors has recently been noted in these systems. The present study has partially characterized a soluble suppressive activity extracted from the thymus glands of chronically infected mice (SmTsF) that modulates granuloma formation in acutely infected mice. The suppressive effect of SmTsF could be administered by multiple i.v. injections or by slow release from osmotic minipumps implanted i.p. Homologous and reciprocal transfers of SmTsF prepared from B10.A(3R) and B10.A(5R) donors indicated that SmTsF-induced suppression required homology between the donor and recipient at the I-J subregion of the major histocompatibility complex. Furthermore, the use of immunoabsorbents prepared with anti-I-Jk and anti-I-Jb sera demonstrated that CBA/J (H-2k) SmTsF was retained by, and could be recovered from, anti-I-Jk insoluble columns, but was unaffected by parallel treatment with anti-I-Jb sera. Subsequent immunoabsorbent studies showed that SmTsF did not bind to soluble egg antigenic (SEA) columns, and thus demonstrated a lack of idiotype, anti-antigen activity. However, columns prepared by using anti-SEA IgG from chronically infected syngeneic mice retained SmTsF suppressive activity, and it could be recovered by alkaline elution. These data are compatible with an interpretation that the suppressive activity expressed anti-idiotypic reactivity. Thus a thymus extract obtained from chronic, modulated, S. mansoni-infected mice can induce granuloma suppression in acutely infected mice. This activity is associated with an I-J determinant-bearing, possibly anti-idiotypic moiety or moieties. These observations further implicate some of the Ts cascades reported in other systems in the regulation of cell-mediated pathogenesis in chronic experimental schistosomiasis.     

The molecular biology of schistosomes

Investigators in various laboratories have been studying the molecular biology of schistosome genetics for several years. A recent meeting at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA, sponsored by the Edna McConnell Clark Foundation, brought together 30 scientists from 16 laboratories to share what they have learned from their studies and debate strategies and techniques.     

Increased phospholipid synthesis in the stimulated rat and human pancreas

Stimulation of the exocrine pancreas is associated with marked changes in pancreatic phospholipid metabolism. It has been previously established that de novo synthesis of phospholipids constitutes part of this "phospholipid effect". This study has demonstrated that in vitro stimulation of the rat pancreas utilising bethanecol and pancreozymin results in increased incorporation of labelled glucose into phosphatidyl inositol and, to a lesser extent, other phospholipids, suggesting increased de novo synthesis of these compounds. However, secretin which is believed to act via a different intracellular pathway, did not exert such an effect. The relevance of this animal model is indicated by the demonstration of increased incorporation of labelled glucose into phospholipids of human pancreas stimulated in vitro by bethanecol or sincalide (the active carboxy terminal octapeptide of pancreozymin).     

Selectivity in phagocytosis and persistence of symbiotic algae in the scyphistoma stage of the jellyfish Cassiopeia xamachana

We have investigated whether interactions between cell-surface macromolecules play a role in cellular recognition leading to specificity in the establishment of intracellular symbiosis between dinoflagellates and the polyp (scyphistoma) stage of the jellyfish Cassiopeia xamachana. All strains of the symbiotic dinoflagellate Symbiodinium microadriaticum were phagocytosed by the endodermal cells of the scyphistomae when presented to them as cells freshly isolated from their respective hosts. The rates of phagocytosis of such cells were high, and were directly correlated with the presence of a membrane, thought to be the host cell vacuolar membrane that surrounds the freshly isolated algae. Cultured algae lack this membrane. All cultured algae, even those that proliferate in host tissues, were phagocytosed at very low or undetectable rates. Freshly isolated algae treated with reagents that removed the host membrane were phagocytosed at low rates. The endodermal cells of the scyphistomae of the non-symbiotic medusa Aurelia aurita also phagocytosed freshly isolated algae, but did not phagocytose cultured algae. Phagocytosis of algae and carmine particles was found to be a competitive process in scyphistomae of C. xamachana. No correlation was observed between the surface electrical charge on algae and their phagocytosis by host endodermal cells. Neither was there any correlation between phagocytosis and persistence. We conclude that the specificity in symbioses between marine invertebrates and dinoflagellates appears to be regulated by processes that occur after potential algal symbionts are phagocytosed.     

Expression of cross-reactive, shared idiotypes on anti-SEA antibodies from humans and mice with schistosomiasis

Immunoaffinity-purified antibodies against Schistosoma mansoni soluble egg Ag (SEA) from infected patients' sera differ idiotypically according to the donor's clinical form of the disease. The Id differ both by their ability to stimulate proliferation of anti-Id T cells and their recognition by anti-Id-specific sera. Also, mice infected with S. mansoni develop anti-Id T and B cell responses against mouse anti-SEA antibodies. We now show that anti-SEA antibodies from serum pools of chronic, but asymptomatic patients (AM1 and AM5) stimulate proliferation of spleen cells from mice infected with S. mansoni. However, AM8, anti-SEA antibodies from hepatosplenic patients, did not stimulate these spleen cells. The murine responses directly parallel patient studies where AM1 and AM5 Id-stimulated human PBMC, but AM8 Id did not. In competitive ELISA, using AM1 or AM-5-specific rabbit antisera or human anti-SEA mAb E5-specific rabbit antiserum, sera from mice infected for 8 and 16 wk (but not from uninfected mice) compete with AM1, AM5, or E5. These sera do not compete in the AM8/anti-AM8 competitive ELISA. Sera from 8-wk-infected mice inhibit more against AM1, AM5, and E5 than do sera from later infections, and anti-SEA immunoaffinity-purified antibodies from 8-wk-infected mice stimulate spleen cells from infected mice more than anti-SEA antibodies from sera of mice late in infection. However, spleen cells from more chronically infected mice are more responsive to either the murine or human anti-SEA antibody preparations than cells from mice with earlier infections. Both the ELISA data and lymphocyte responses indicate that anti-SEA antibodies from mice infected with S. mansoni for 8 wk bear Id cross-reactive with those expressed on anti-SEA antibodies from humans with chronic, asymptomatic schistosomiasis, but not those from hepatosplenic patients.     

IMPRINTING EFFECTS OF THREE AMINO ACIDS (ALANINE,LYSINE AND GLYCINE) AND THEIR OLIGOPEPTIDES INTETRAHYMENA PYRIFORMIS. DATA FROM THE HORMONE AND HORMONE RECEPTOR EVOLUTION

Hormonal imprinting takes place at the first encounter of the hormone and receptor, and results in a changed binding capacity and reaction of the cell and its progeny generations. The imprinting effect of three amino acids and their oligopeptides is studied using fluorescent-labelled peptides. Glycine and lysine could provoke positive imprinting (increased binding in the progeny generations) for their own peptides, but alanine could not. Mostly positive imprinting was provoked by glycine and lysine peptides for their own peptides of different chain length. The optimal chain length provoking self-imprinting was four for glycine, two for lysine and three for alanine. Except in this case, alanine was neutral or provoked mostly negative imprinting. After reaching the optimal chain length, there is a decline in binding. Evolutionary conclusions are discussed.