The epithelium overlying rabbit bronchus-associated lymphoid tissue does not express the secretory component of immunoglobulin A

Summary The epithelium associated with lymphoid aggregates in the bronchial tract (BALT) was studied in rabbits by immunohistochemistry using monoclonal antibodies against the secretory component (SC) of IgA. The normal bronchus epithelium was intensely labelled. In contrast, epithelium overlying the central parts of the follicles was negative. This specialized epithelium cannot participate in the SC-mediated transport of IgA, which might be a basis for the adherence and transport of microorganisms into the lymphoid tissue, thus initiating immune responses of the BALT.     

Inhibition of acrosin by oviductal secretory immunoglobulin A

A major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α-chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.     

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity

The protozoan Entamoeba histolytica is the etiological agent of amebiasis, an infection with high prevalence worldwide. The host-ameba relationship outcome depends on parasite and host factors, and among these is secretory IgA. These antibodies reduce mucosal colonization by pathogens and neutralize a variety of toxins and enzymes. The functionality of secretory IgA depends on its integrity. Some bacteria produce IgA proteases that cleave mainly the IgA1 subclass; live E. histolytica trophozoites, and other ameba fractions are also able to degrade human IgA. The aim of this study was to determine if serum and secretory IgA, its subclasses and secretory component, are degraded by cysteine proteases, which are present and active on the surface of glutaraldehyde-fixed amebas. It was observed that secretory IgA1, IgA2, free and IgA-bound secretory component were degraded by E. histolytica surface-associated cysteine proteinases. Secretory IgA2, although it was degraded, conserved its ability to agglutinate live amebas better than IgA1. Therefore, while specificity of known ameba cysteine proteases is cathepsin B-like and is different from bacterial IgA proteases, IgA2 was functionally more resistant than IgA1 to ameba surface-associated cysteine protease degradation, similar to the greater resistance of IgA2 to bacterial IgA-specific proteases.     

Toll‐like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria

Toll‐like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin‐specific humoral immunity. Further, TLR5‐expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)‐specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5^?/?) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella‐Tox C), tetanus toxoid (TT)‐ and flagellin (FliC)‐specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT‐specific IgG Ab‐forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5^?/? mice were comparable to those in wild‐type mice. rSalmonella‐Tox C was equally disseminated in TLR5^?/? mice, TLR5^?/? mice lacking Peyer's patches (PPs), and wild‐type mice. In contrast, TLR5^?/? PP‐null mice failed to induce TT‐ and FliC‐specific SIgA Abs in the intestine and showed significantly reduced numbers of TT‐specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag‐specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.

     

Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

Abstract The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobullin A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421. This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release. The anti-bacterial activity of lactoferrin was not affected by protease treatment. Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. The susceptibility of secretory immunoglobulin A to V. cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.     

A molecular chaperone mediates a two-protein enzyme complex and glycosylation of serine-rich streptococcal adhesins

Serine-rich repeat glycoproteins identified from streptococci and staphylococci are important for bacterial adhesion and biofilm formation. Two putative glycosyltransferases, Gtf1 and Gtf2, from Streptococcus parasanguinis form a two-protein enzyme complex that is required for glycosylation of a serine-rich repeat adhesin, Fap1. Gtf1 is a glycosyltransferase; however, the function of Gtf2 is unknown. Here, we demonstrate that Gtf2 enhances the enzymatic activity of Gtf1 by its chaperone-like property. Gtf2 interacted with Gtf1, mediated the subcellular localization of Gtf1, and stabilized Gtf1. Deletion of invariable amino acid residues in a conserved domain of unknown function (DUF1975) at the N terminus of Gtf2 had a greater impact on Fap1 glycosylation than deletion of the C-terminal non-DUF1975 residues. The DUF1975 deletions concurrently reduced the interaction between Gtf1 and Gtf2, altered the subcellular localization of Gtf1, and destabilized Gtf1, suggesting that DUF1975 is crucial for the chaperone activity of Gtf2. Homologous GtfA and GtfB from Streptococcus agalactiae rescued the glycosylation defect in the gtf1gtf2 mutant; like Gtf2, GtfB also possesses chaperone-like activity. Taken together, our studies suggest that Gtf2 and its homologs possess the conserved molecular chaperone activity that mediates protein glycosylation of bacterial adhesins.     

Identification and Characterization of a Novel Prokaryotic Peptide: N-GLYCOSIDASE FROM ELIZABETHKINGIA MENINGOSEPTICA*

Peptide:N-glycosidase (PNGase) F, the first PNGase identified in prokaryotic cells, catalyzes the removal of intact asparagine-linked oligosaccharide chains from glycoproteins and/or glycopeptides. Since its discovery in 1984, PNGase F has remained as the sole prokaryotic PNGase. Recently, a novel gene encoding a protein with a predicted PNGase domain was identified from a clinical isolate of Elizabethkingia meningoseptica. In this study, the candidate protein was expressed in vitro and was subjected to biochemical and structural analyses. The results revealed that it possesses PNGase activity and has substrate specificity different from that of PNGase F. The crystal structure of the protein was determined at 1.9 Å resolution. Structural comparison with PNGase F revealed a relatively larger glycan-binding groove in the catalytic domain and an additional bowl-like domain with unknown function at the N terminus of the candidate protein. These structural and functional analyses indicated that the candidate protein is a novel prokaryotic N-glycosidase. The protein has been named PNGase F-II.     

Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM₁, as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis.     

Relationship between canine mucosal and serum immunoglobulin A (IgA) concentrations: serum IgA does not assess duodenal secretory IgA

A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.     

Delivery of ferric ion to mouse spermatozoa is mediated by lipocalin internalization

The aim of this study was to illustrate the further process of 24p3 protein after association with epididymal spermatozoa. We have previously identified a caput-initiated 24p3 protein, which interacts with the spermatozoa surface in vitro. In the present study, we investigate another role of the 24p3 protein with spermatozoa. Mouse epididymal spermatozoa exhibit the ability to bind spontaneously with exogenous 24p3 protein, a part of which is further internalized into the spermatozoa in epididymal caput. We have now focused on this issue using freshly prepared spermatozoa from caudal region of epididymis. First, the cytosolic fractionation of spermatozoa has revealed that biotinylated 24p3 protein signal could be detected by supplying biotinylated protein under 37 degrees C incubation after 30 min at this experiment. Further, flow cytometric analysis of FITC-protein containing spermatozoa has revealed two distinct types of fluorescent spermatozoa, and microscopical experimentation with fluorescent FITC-24p3 protein has shown that the 24p3 protein did accumulate in the cytosolic portion of spermatozoa. All of these events, which showed protein uptake into the cell, demonstrated time- and temperature-dependence of endocytotic characteristics, these constituting the critical points in the process of endocytosis for spermatozoa as for other cells. Using a fluorometric method, the binding affinities of ferrous ion and ferric ion to 24p3 protein were shown to be (1.5+/-0.2)x10(6) and (3.0+/-0.4)x10(7)M(-1), respectively. We have also determined the internalization of this protein in the transition of iron into spermatozoa. We report here that spermatozoa, from the caudal epididymis, demonstrate the ability to bind with 24p3 protein and further internalize it and deliver the ferric ion to the spermatozoa via protein internalization. We suggest that the 24p3 protein plays a physiological role in spermatozoa in the context of protein-ligand complex internalization.     

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer''s patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c⁺CD11b⁺CD8⁻ dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.     

A significant fraction of calcium transients in intact Guinea Pig ventricular myocytes is mediated by Na-Ca exchange

Ca²⁺ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca²⁺ changes across the cell, suggesting the presence of significant spatial Ca²⁺ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca²⁺ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca²⁺]_i elicited by membrane depolarization. The overall spatial distribution of [Ca²⁺]_i changes appeared unchanged. Ca²⁺ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the exchange mechanism. Conversely, when the reversal potential of the exchange was shifted to negative potentials by lowering [Na⁺]₀ or by increasing [Na⁺]_i by treatment with 20 μM monensin, the amplitude of these Ca²⁺ transients increased. Ca²⁺ transients elicited by membrane depolarization and largely mediated by reverse operation of Na⁺-Ca²⁺ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca²⁺ influx through the exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.     

A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' beta-mannoside linkage. This linkage was installed by forming initially the corresponding beta-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the beta-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a beta-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated alpha-glycosyl chlorides with thioacetate to give the peracetylated beta-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.     

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.     

Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.     

Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galβ1–3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the β-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the β-strands, requiring them to bend to match the concavity of the VLR solenoid.     

Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.     

Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.