Biosynthesis and turnover of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

Antiserum was raised in rabbits against a bile canalicular glycoprotein of Mr = 110,000 purified to homogeneity from of rat liver. The antisera specifically immunoprecipitated a Mr = 110,000 polypeptide from hepatocytes metabolically labeled with [35S]methionine. When hepatocytes in primary culture were incubated with tunicamycin before labeling with [35S]methionine in the presence of tunicamycin, the major polypeptide immunoprecipitated by the specific antiserum from Triton X-100 extracts of cells had a molecular weight of 59,000. Enzymatic removal of N-linked carbohydrates from the Mr = 110,000 glycoprotein by N-glycanase digestion also yielded a polypeptide with minimum Mr = 59,000. In pulse-chase experiments using [35S]methionine, the Mr = 110,000 protein detected by the specific antisera first appears as Mr = 85,000 and 75,000 intermediate species which are endoglycosidase H sensitive. The Mr = 85,000 intermediate form is lost first with time followed by the Mr = 75,000 form giving rise to the Mr = 110,000 form that is endoglycosidase H resistant. Neuraminidase digestion of the Mr = 110,000 form generated an Mr 85,000 form but with a different carbohydrate structure than the intermediate Mr 85,000 form detected in the pulse-chase experiments. The time required to accomplish the processing of the Mr = 85,000 and 75,000 forms is relatively slow. Finally, the terminal sugars are added and the mature Mr = 110,000 glycoprotein is rapidly transported to the cell surface. A minimum time of 90 min is required for the Mr = 110,000 bile canalicular glycoprotein to be synthesized, processed, and reach the cell surface which is long relative to the time required (10 min) for another domain-specific protein, the receptor for asialoglycoproteins, to reach the sinusoidal surface. The Mr = 110,000 bile canalicular glycoprotein turns over in the bile canalicular domain with a half-life of 43 h while the asialoglycoprotein receptor turns over in the sinusoidal domain with a half-life of 23 h.     

Resistance of native, oligomeric envelope on simian immunodeficiency virus to digestion by glycosidases

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-beta-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and beta-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-beta-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.     

The cell wall of Chlamydomonas eugametos. Immunological aspects

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.     

The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus.

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membrane-bound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipitated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83- to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N-glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal microscopy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.     

Fatty acylation of yeast glycoproteins proceeds independently of N-linked glycosylation

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild-type and asparagine-linked glycosylation-deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non-permissive temperature (37 degrees C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS-PAGE. In contrast, the wild-type yeast cells contained [3H]palmitate-labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS-PAGE. In addition, labeling of the wild-type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non-permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N-glycosylation in yeast cells.     

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.     

Biochemical,immunological, and structural studies on a sphingolipid activator protein (SAP-1)

Sphingolipid activator protein-1 (SAP-1) is a glycoprotein found in human tissue extracts that stimulates the enzymatic hydrolysis of at least two glycosphingolipids, including GM1 ganglioside and sulfatide. The ability of purified SAP-1 to stimulate GM1 ganglioside hydrolysis by extracts of cultured fibroblasts from patients with β-galactosidase deficiency was examined, and all patients had a pronounced deficiency (under 10% of control). Using monospecific antibodies against SAP-1, the concentration was determined in cultured fibroblasts by rocket immunoelectrophoresis. Extracts from 15 control cell lines were found to have 0.72 ± 0.24 μg cross-reactive material/mg protein, while cell extracts from 8 patients with GM1 gangliosidosis involving mental retardation were found to have 1.08 ± 0.17, which is significantly elevated. When the fibroblast extracts were subjected to sodium dodecyl sulfate-polyacramide gel electrophoresis followed by electroblotting, multiple bands were observed. Controls were found to have two major bands with estimated molecular weights of 9000 and 9500, and a minor band at 7800. Extracts from patients with GM1 gangliosidosis were found to have multiple bands ranging upward to 13,000. Extracts from patients with the most severe clinical types of GM1 gangliosidosis had almost exclusively high-molecular-weight forms (molecular weights above 10,000). Treatment of SAP-1 from control liver with endoglycosidase D caused a decrease in the M_r 9500 band and increased in the M_r 7800 band. When SAP-1 from GM1 gangliosidosis liver was treated sequentially with neuraminidase, β-galactosidase, and endoglycosidase D, almost all of it was converted to the forms found in control human liver.     

The UL10 gene of herpes simplex virus 1 encodes a novel viral glycoprotein, gM, which is present in the virion and in the plasma membrane of infected cells.

The herpes simplex virus 1 UL10 gene encodes a hydrophobic membrane protein dispensable for viral replication in cell culture (J.D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). We report the following. (i) A fusion protein consisting of glutathione S-transferase fused to the C-terminal 93 amino acids of the UL10 protein was used to produce a rabbit polyclonal antiserum. The antiserum reacted with infected-cell proteins which formed in denaturing polyacrylamide gels a sharp band (apparent M(r) of 50,000) and a very broad band (M(r) of 53,000 to 63,000). These bands were not formed by lysates of UL10- virus or by lysates of infected cells boiled in the presence of sodium dodecyl sulfate before electrophoresis. (ii) The proteins forming both bands were labeled by [3H]glucosamine, indicating that they were glycosylated. (iii) The UL10 protein in cells treated with tunicamycin formed a single band (apparent M(r) of 47,000) reactive with the anti-UL10 antibody, indicating that the 47,000-M(r) protein was a precursor of N-glycosylated, more slowly migrating forms of UL10. Treatment of the immunoprecipitate with endoglycosidase H increased the electrophoretic mobility of the 50,000-M(r) species to that of the 47,000-M(r) species, indicating that the 50,000-M(r) species contained high-mannose polysaccharide chains, whereas the proteins forming the 53,000- to 63,000-M(r) bands contained mature chains inasmuch as they were resistant to digestion by the enzyme. (iv) The UL10 protein of R7221 carrying a 20-amino-acid epitope formed only one band with an M(r) of 53,000. This band was sensitive to endoglycosidase H, suggesting that the epitope inserted in the R7221 UL10 protein may have interfered with glycosylation. (v) The UL10 protein does not contain a cleavable signal sequence inasmuch as the first UL10 methionine codon was reflected in the 50,000-M(r) protein. (vi) The UL10 protein is present in virions and plasma membranes of unfixed cells that were reacted with the polyclonal rabbit antibody. In accordance with the current nomenclature, the UL10 protein is designated glycoprotein M.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

In vitro sulfation of pulmonary surfactant-associated protein-35

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.     

Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus.

Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article).     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Polyadenylated RNA prepared from first trimester human placenta was translated in a membrane-free cell-free system derived from wheat germ. Analysis of the [³⁵S]methionine-labeled products by SDS-polyacrylamide electrophoresis demonstrated two proteins with apparent M_rs of 14,500 and 16,000 that were specifically immunoprecipitated by antiserum to reduced and carboxylated bovine LH_α, and two different proteins with apparent M_rs of 18,500 and 21,000 that were specifically immunoprecipitated by antiserum to hCG_β. None of these products was sensitive to cleavage by endoglycosidase H, whereas the M_r 21,000 product precipitated by antisera to bovine LH_α and to hCG_α from translations supplemented by canine pancreatic microsomes was processed to a product with M_r 13,000 by endoglycosidase H. We suggest that the two forms of the α and β subunit precursors could arise from the translation of two distinct mRNAs encoding each subunit.     

Synthesis and processing of cellulase from ripening avocado fruit

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.     

Evidence that leukosialin, CD43, is intensely sulfated in the murine T lymphoma line RDM-4.

Metabolic labeling of the murine T lymphoma cell line RDM-4 with [35S] sulfate results in intense incorporation into a cell-retained component of apparent Mr approximately 100,000. This macromolecule is identified as a glycoprotein by lectin chromatography. The sulfate is not incorporated as tyrosine sulfate. Release of the radiolabel by alkaline beta-elimination but not by endoglycosidase F is consistent with the sulfation of O- rather than N-linked oligosaccharides. The sulfated glycoprotein displays anomalous migration on SDS-PAGE in two respects: 1) the apparent Mr shifts from 115,000 to 87,000 on increasing the acrylamide concentration from 7 to 12%, and 2) on neuraminidase digestion migration is substantially reduced (apparent Mr 140,000). These properties indicate that the sulfated protein is both heavily glycosylated and extensively sialylated, and are characteristic of the lymphoid mucin, leukosialin (sialophorin, CD43). Specific labeling of the sialoglycoproteins of RDM-4 cells indicates that leukosialin, the most intensely labeled protein, comigrates with the sulfated protein on SDS-PAGE at varying acrylamide concentrations. Our data are therefore consistent with sulfation of at least some of the numerous O-linked oligosaccharides of this abundant glycoprotein in RDM-4 cells. No sulfation of CD43 in resting splenic T cells is observed.     

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.     

Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

Surface changes in chimpanzee sperm during epididymal transit

Intact chimpanzee caput and cauda epididymal sperm, sperm cell lysates, and caput and cauda epididymal fluid were radiolabeled by enzymatic iodination with lactoperoxidase and Na125 I and were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Caput epididymal sperm showed nine labeled macromolecular components of 90, 64, 56, 48, 38, 31, 20, 18 and 16 Kd and cauda epididymal sperm showed eleven macromolecular components of 90, 64, 55, 47, 42, 33, 27, 18, 17, 15 and 11 Kd. Six of the components labeled on caput sperm (90, 64, 56, 48, 18 and 16 Kd) were detected in equal amounts of cauda sperm and two (38 and 20 Kd) were detected at greatly reduced labeling intensities. In the cauda epididymidis, four new components (33, 27, 17 and 11 Kd) became prominent features of the sperm surface. Analysis of labeled caput and cauda sperm cell lysates resolved components distinct from those detected on sperm surfaces. Electrophoresis of caput epididymal fluid showed five labeled components of 66, 56, 47, 41 and 37 Kd, while electrophoresis of cauda epididymal fluid showed eight labeled components of 92, 66, 56, 48, 31, 27, 24 and 11 Kd. Three components (66, 56 and 47 Kd) were present in both caput and cauda fluid, two (41 and 37 Kd) in caput fluid only, and five (92, 31, 27, 24 and 11 Kd) in cauda fluid only. Components of 37 Kd were labeled in caput fluid and on caput sperm but not on cauda sperm, whereas components of 27 Kd and 11 Kd were labeled in cauda fluid and on cauda sperm but not on caput sperm. These data show that chimpanzee sperm undergo extensive surface modifications during epididymal maturation and that some of these modifications may be related to exogenous proteins/glycoproteins in epididymal fluids.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.