Yolk hydrolase activities associated with polypeptide and oligosaccharide processing of Blattella germanica vitellin

Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among the subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, the M_r 160,000 subunit Vt is cleaved to products of M_r 95,000 and M_r 50,000. In association with an unprocessed M_r 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 30°C), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4–5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event. Although the predominant oligosaccharide of Vt is Man₉GlCNAc₂, the M_r 50,000 subunit of egg-borne Vt contains a much higher proportion of Man₆GlCNAc₂ than the other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an oothecapermits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-α-mannosidase, exp-β-N-acetylglucosaminidase, α-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-β-N-acetyl-glucosaminidase activities coincide with the day 4–5 proteolysis, while α-mannosidase remains relatively constant throughout the first 6 days. Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.     

The occurrence of artemocyanin in Branchiopoda (Crustacea)

Artemocyanin, the extracellular hemolymph biliprotein of Artemia, is demonstrated in the fairy shrimp Streptocephalus, the clam shrimp Leptestheria and the water flea Daphnia. Artemocyanins can be purified from hemolymph as intact polypeptides (M_r 170–190,000), but are degraded upon homogenization of the whole animal by partial proteolysis to polypeptides with M_r 102,000 and 85,000. The aminoterminal sequence of the intact artemocyanin polypeptide was determined, but no clear-cut relationships with arthropod biliproteins or other protein families could be demonstrated.     

Proteolytic processing of Blattella germanica vitellin during early embryo development

In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization is initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt's processing, its specific activity with synthetic substrates increased four-fold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt's large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (B) processed the M_r 102,000 subunit but not the M_r 95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant's eggs at day 6 mirrored the extent of processing, being highest in group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It is concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic examination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diameters of 15–30 μm to 3–10 μm. Yolk granules of those translocant's eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally.     

Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons

-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (M_r) 66000 and M_r 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. -Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an M_r of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the M_r-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that -mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [³H]glucosamine shows that after processing only the M_r-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to -mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of -mannosidase out of the ER-Golgi system.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - H L heavy, light subunit - IgG Immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The effect of periodate oxidation and α-mannosidase treatment of Dolichos biflorus lectin

The effects of periodate and α-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lactin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or α-mannosidase rendered the lecton nonprecipitable by concanavalin A. The lectrin treated by α-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity.The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or α-mannosidase treatment.     

The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins

α-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The α-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified α-mannosidase activity depends on a single Zn²⁺ ion per subunit is inhibited by swainsonine with an IC₅₀ of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(1,6)-d-mannobioses. In addition, the enzyme is able to demannosylate Man₃GlcNAc₂ and Man₇GlcNAc₂ oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ssα-man removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins.     

Comparison of α-glucosidase and α-mannosidase in bloodstream forms of Trypanosoma brucei brucei

• 1.1. The physicochemical and kinetic properties of the two major trypanosomal glycosidases, α-glucosidase (EC 3.2.1.20) and α-mannosidase (EC 3.2.1.24), were compared in bloodstream forms of Trypanosoma brucei brucei S42.
• 2.2. Both enzymes are membrane-bound and located intracellularly.
• 3.3. The results are discussed in relation to the possible role of α-glucosidase and α-mannosidase in the processing or catabolism of trypanosomal glycoproteins.

     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Nascent α2-macroglobulin-trypsin complex: Incorporation of amines and water at the thiol esterified GLX-residues of α2-macroglobulin during activation with trypsin

During complex formation between α₂-macroglobulin and trypsin the internal thiol esters (one in each of the four M_r 180,000 subunits) are activated. In this activated state (nascent α₂-macroglobulin-trypsin complex) addition of low M_r amines lead to their covalent incorporation into α₂M. Evidence is presented showing that covalent binding of added amines occurs at the γ-carbonyl group of the Glx-residue in the thiol ester sequence:

     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells

Leukimia-associated inhibitory activity suppresses colony and cluster formation in vitro of cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. It was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by α-methylmannose from concanavalin A-Sepharose columns and had an apparent M_r range of 450–550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.     

Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with α-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean α-mannosidase. (d) Treatment of the native proteins with α-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all α-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized α-mannosidase.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

α-Mannosidase from jack bean (Canavalia Ensiformis): Negative staining of single molecules and paracrystalline forms

Purified jack bean α-mannosidase has been shown by conventional negative staining, using sodium phosphotungstate, to be a dimeric molecule, length ca 11.5 nm and width ca 3 nm. Electrophoretic data suggests that the dimeric molecule has a M_r of ca 400,000 and that it contains two subunits of M_r ca 135,000. It is proposed that the stable dimeric state of the molecule has the conformation (αβ)₂. Trace quantities of a tetrameric form are, however, detected by both electron microscopy and electrophoresis. Attempts to crystallize α-mannosidase in the presence of ethanol have led to the production of irregular molecular aggregates of varying size, but prolonged storage in the presence of 80% (v/v) ethanol produced some aggregates which exhibit quasi-crystallinity. The instability of the crystalline particles during the negative staining procedure presents a problem. Application of the negative staining-carbon film technique has been found to produce ca 10 nm rodlike structures, which tend to be oriented randomly, relative to one another. However, if polyethylene glycol-1000 (0.05%) is included before the mixture of enzyme and ammonium molybdate is allowed to dry onto the mica surface, the formation of two-dimensional paracrystals composed of a number of parallel 10 nm rods is induced. Under these conditions the 10 nm rods are shown to have a greater amount of negative stain located in a central channel than is the case in the absence of polyethylene glycol-1000.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Occurrence of glycoprotein glycosidases in mature seeds of mung bean (vigna radiata)

The presence of nine different glycosidases was demonstrated in the crude extract of mature mung bean seeds. N-Acetyl β-D-glucosaminidase, α-D-galactosidase and β-D-glucosidase were each resolved into two respective active forms by gel filtration. The other glycosidases showed single forms only. The apparent MWs of the glycosidases were determined. The glycosidases were absorbed to Con A-Sepharose column, with the exception of a small percentage of α-galactosidase and α-mannosidase which were eluted unretarded. The bound enzymes displayed varying affinities for the immobilized lectin, indicating differences in glycosylation. With the exception of β-galactosidase and invertase, all the glycosidase activities were detected in the protein bodies isolated from the seeds.     

Characterization of antifungal glycoprotein in red-light-irradiated broadbean leaflets

 Red-light treatment of broadbean leaflets resulted in the production of antifungal substance(s) against Botrytis cinerea. The antifungal substance(s) was positively charged, as the antifungal constituent was removed by the cation exchanger CM cellulose. Treatment of infection droplets with glycosidases (α-mannosidase, β-galactosidase, β-glucosidase), glycol-specific reagent periodate (NaIO₄), and proteinase K completely eliminated antifungal activity, suggesting that both protein and carbohydrate are active components. The protein content of infection droplets was 0.148 mg/ml. The HPLC gel column analysis of infection droplets resulted in four fractions; all the fractions showed antifungal activity. Received: June 14, 2002 / Accepted: August 12, 2002 Correspondence to:Y. Honda     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Studies on binding and uptake of vitellogenin by follicles of the tobacco hornworm,Manduca sexta

An in vitro system for the uptake of ¹²⁵l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with ¹²⁵l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that ¹²⁵l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, ¹²⁵l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (K_D ? 1.3 × 10^?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 10¹⁴ sites/g of membrane protein. Competition studies showed that binding of ¹²⁵l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (M_r ～ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (M_r ～ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.