Homology of the D-galactose-specific lectins from Artocarpus integrifolia and Maclura pomifera and the role of an unusual small polypeptide subunit

The Maclura pomifera agglutinin (MPA) was purified by affinity chromatography from a seed extract and its properties were compared with those of the Artocarpus integrifolia lectin, jacalin. Reverse-phase high-performance liquid chromatography showed both proteins had multiple forms of a small approximately 20-residue polypeptide chain in addition to the major 12,000 Mr subunit. The amino acid sequences of the small chains and the N-terminal sequences of the large subunits showed considerable similarity between the two proteins, approximately 60% identical residues. The homology of the proteins was confirmed by the similarity of their circular dichroism and fluorescence emission spectra. MPA showed much greater spectral changes upon binding methyl alpha-D-galactoside, suggesting it has complete activity rather than the partial activity found for jacalin. The binding of methyl alpha-D-galactoside by MPA was measured by fluorescence titration; the KA was 1.9 X 10(4) M-1 compared to 3.4 X 10(4) M-1 for jacalin. MPA also precipitated human IgA1 in the same manner as jacalin. The spectra indicate the involvement of tryptophan and tyrosine residues in the binding site of these lectins. Since a tryptophan residue is conserved in all the small subunits, they may form part of the binding site.     

Binding of IgA by <Emphasis Type="Italic">Mycoplasma penetrans</Emphasis>

The current study shows that Mycoplasma penetrans strain GTU binds human serum immunoglobulin A (IgA) and secretory IgA but not IgG. Binding of IgA was associated almost exclusively with the lipoprotein fraction obtained by Triton X-114 fractionation of isolated M. penetrans membranes. Western immunoblot analysis of isolated membranes of M. penetrans strain GTU and of the Triton X-114 fraction showed that the major IgA-binding component was a lipoprotein with a molecular mass of 38 kDa, one of the major lipoproteins of this organism. The authors suggest that the high IgA-binding capacity of M. penetrans strain GTU may serve as a defense mechanism, conferring on this microorganism the ability to evade clearance mediated by specific IgA antibodies.     

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.     

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

The preprotein translocase of the outer mitochondrial membrane (also called TOM complex) from Arabidopsis thaliana was characterized by Blue-native gel electrophoresis (BN-PAGE) and Electrospray Tandem Mass Spectrometry (ESI-MS/MS). BN-PAGE allows to prepare a very stable 390 kDa complex that includes six different protein types: the 34 kDa translocation pore TOM40, the 21/23 kDa preprotein receptor TOM20, the small TOM component TOM7 and three further subunits of 10, 6.3 and 6.0 kDa. Primary structures of all TOM subunits were elucidated. The 10 kDa subunit represents a truncated version of the TOM22 preprotein receptor and the two 6 kDa proteins represent subunits possibly homologous to fungal TOM6 and TOM5, although sequence conservation is at the borderline of significance. TOM40, TOM7 and one or both of the 6 kDa subunits form a subcomplex of about 100 kDa. The six TOM proteins from Arabidopsis are encoded by 12 genes, at least 11 of which are expressed. While the subunit composition of the TOM complex from fungi, animals and plants is remarkably conserved, the domain structure of individual TOM proteins differs, e.g. acidic domains in TOM22 and the 6 kDa TOM subunits from Arabidopsis are absent. The domain structure of the Arabidopsis TOM complex does not support the so-called ‘acid chain hypothesis’, which explains the translocation of proteins across the outer mitochondrial membrane of mitochondria by the binding of preproteins to acidic protein domains within the TOM complex. Functional implications are discussed.     

In vitro proteolytic processing of pea and jack bean storage proteins by an endopeptidase from lupin seeds

The highly specific proteolytic breakdown observed upon prolonged treatment of pea legumin and pea and jack bean vicilin with a thiol endopeptidase purified from mature lupin seeds has been studied in detail. Proteolytic cleavage occurred in the acidic subunits of pea legumin, whereas the basic subunits were unaffected. Jack bean vicilin (M, 47 K) was cleaved near the middle of the polypeptide chain, whereas pea vicilin (M, 50 K) was cleaved into two fragments of M, 30 K and 20 K, respectively. The 30 K M, polypeptide chain contained covalently linked carbohydrate and had an N-terminal sequence suggesting that cleavage had taken place between the α and β region of the vicilin 50 K M, polypeptide as previously described in vivo. These results suggested that the cleavage specificity of lupin endopeptidase was in the proximity of paired arginine amino acid residues.The changes in the vicilin polypeptides due to proteolytic cleavage by lupin enzyme and those occurring during germination of pea seeds are also reported and discussed.     

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.     

Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect’s digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 ??M). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.     

Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue

The nature of IgA-binding cells and their tissue distribution was examined by an indirect immunofluorescence assay with the use of IgA1 and IgA2 paraproteins and fluorochrome- or biotin-labeled F(ab')2 fragments of idiotype-specific antibodies. The frequency of IgA-binding mononuclear cells was approximately 13% in blood and spleen samples but less than 1% in tonsil samples. IgA binding could be visualized by flow immunocytometry on monocyte/macrophages, but not on T and B cells. IgA polymers were bound better than IgA dimers and monomers. Nonhomologous IgA myelomas of both IgA1 and IgA2 subclasses inhibited the IgA-binding to monocytes, whereas aggregated normal serum IgG, IgM paraproteins, and an IgG myeloma did not. IgA binding was relatively insensitive to changes in temperature or cation concentration. IgA-binding monocytes were found in IgA-deficient patients at the same frequency as in normal individuals. The results indicate that monocytes constitutively express class-specific binding sites for both IgA1 and IgA2 molecules.     

Characterization of human arylsulfatase a glycans

Despite numerous studies on arylsulfatase A, the structure of the glycans present in each of its two subunits has not been determined. This is important because the carbohydrate component of human arylsulfatase A synthesized in tumor tissues and transformed cells has been shown to undergo apparent changes. This study elucidates some of their major features.Glycan chain analysis of native and deglycosylated arylsulfatase A as well as its subunits was performed with the use of a Glycan Differentiation Kit and lectin affinity chromatography. Each of the two subunits of arylsulfatase A from placenta, separated electrophoretically on polyacrylamide gel in reducing conditions, reacted with digoxigenin-labelled Galantus nivalis agglutinin and Aleuria awantia agglutinin, while those from liver enzyme reacted with the former only. The subunits of both enzymes did not react with Sambucus nigra, Maakia amuriensis, Datura stramonium or Peanut agglutinin. Deglycosylation of arylsulfatase A with peptide N-glycosidase F and endo-β-N-acetylglucosaminidase F resulted in complete cleavage of its carbohydrate component from each subunit. Their molecular weights decreased by 3 kDa. Neuraminidase treatment of the enzyme from liver and placenta followed by isoelectrofocusing separation showed the presence of sialylated forms which constituted a small percentage of total enzyme activity. Placental arylsulfatase A became bound to Lens culinaris agglutinin agarose, while no interaction with Ricinus communis or Griffonia simplicifolia agglutinin agarose was observed.The study shows that both subunits of arylsulfatase A from human placenta possess two high mannose/hybrid type glycans as major structures, with at least one 6-O-l-fucose bound to the innermost N-acetylglucosamine on each. The enzyme from liver does not possess fucose. Complex type glycans containing sialic acid constitute a small percentage of the total carbohydrate component.     

Ovarian Development and Vitellin of the Mushroom Fly,Lycoriella mali (Diptera: Sciaridae)

Ovarian Development and Vitellin of the Mushroom Fly, Lycoriella mali, were characterized. L. mali has a pair of ovaries, composed of 50–60 ovarioles, respectively. Ovarian development began at 1 day of pupation, and continued to become mature at 2 days after adult emergence. The vitellogenin (Vg) and vitellin (Vn) of L. mali were identified by native- and SDS-PAGE analyses. The Vn is composed of two subunits with a large subunit, L-Vn (190 kDa), and a small subunit, S-Vn (63 kDa). The Vg and Vn detected in the hemolymph and ovary, respectively, have the two equivalent subunits (190, 63 kDa), respectively. These two subunits of Vn gradually decreased in content during embryogenesis. We confirmed the presence of the Vg and Vn which were first detected in the 3 day-old female pupae by SDS-PAGE and Western blot analyses. It can be assumed that the Vn is synthesized as a precursor (Vg) in the fat body at 3 days after pupation and released into hemolymph. Then, the Vg is subsequently absorbed into the ovary at the same time. Twelve amino acid residues after the signal peptide at the N-terminus of L. mali S-Vn were sequenced and showed 70% homology to those of Anthonomus grandis vitellegenin.     

Subunit glycosylation of Lupinus angustifolius seed globulins

Reduced polypeptide subunits of α-, β- and γ-conglutins from Lupinus angustifolius seeds were resolved by preparative SDS gel electrophoresis of the fluorescent labelled proteins, into four, six and two major components, respectively. All subunits were glycosylated, to varying degrees, containing mannose, galactose and glucosamine. The major glycopeptides released by pronase digestion of each conglutin had similar galactose/mannose ratios; the MW of the glycopeptide released from α- and β-conglutin was ca 5000. Although on average, each molecule of α-conglutin contains one main oligosaccharide chain, and β-conglutin two, the presence of carbohydrate in all polypeptide subunits suggests that some subunits may arise by proteolytic cleavage of a larger polypeptide after glycosylation. The presence of minor glycopeptide components indicates that modification of carbohydrate chains during seed development may also occur.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae; Serologic distinction between the receptors by blocking antibodies

Abstract Group A and B streptococci ( Streptococcus pyogenes and Streptococcus agalactiae ) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding epitopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.     

Temperature-sensitive immunoglobulin A-binding and dimerization of C-terminus-impaired protein Arp4 produced in Escherichia coli.

A gene for protein Arp4, an IgA receptor protein derived from Streptococcus pyogenes AP4, was expressed in Escherichia coli. The product was demonstrated to be accumulated in a periplasmic space as a polypeptide with an apparent molecular weight of 40 kDa with the deleted C-terminal membrane anchor portion of protein Arp4. This 40-kDa peptide of the C-terminus-impaired recombinant protein Arp4 produced in E. coli, designated ir-protein Arp4, was purified from a periplasmic fraction of transformants and its IgA-binding activity was analyzed. The IgA binding of ir-protein Arp4 was temperature-sensitive, that is, ir-protein Arp4 bound IgA at 4 and 25 C, but did not at 37 C. In addition, the dimerization of ir-protein Arp4 was also temperature-sensitive in parallel with temperature-dependent binding activity, suggesting that the dimerization of ir-protein Arp4 may be required for its active binding to IgA. In contrast, ir-protein Arp4 immobilized on Sepharose 4B did bind to IgA even at 37 C as well as 4 and 25 C. The immobilized ir-protein Arp4 might acquire the temperature-resistant IgA binding activity in part through the formation of a stable dimerized ir-protein Arp4 on the solid support.