分泌型免疫球蛋白A的研究进展

大部分感染都起源于黏膜表面,而黏膜免疫的主要抗体是分泌型免疫球蛋白A（SIgA）,它能有效地阻断病原体的感染和侵入。SIgA是由1个IgA二聚体、1条J链和1个分泌片（SC）共价结合构成的异源十聚体。IgA和J链由活化B细胞产生,SC则由黏膜上皮细胞合成。SIgA分子具有极高的稳定性和极强的抗微生物活性。我们就SIgA合成的相关机制、IgA单体和SIgA的结构与功能,以及重组SIgA的研究进展简要综述。     

J chain is covalently bound to both monomer subunits in human secretory IgA

Previous work has established that the secretory component (SC) in human secretory IgA is covalently linked to only one of the two IgA monomer subunits, but it has not been clear whether the J chain is covalently linked to one or to both of these subunits. In view of the asymmetry in the disulfide bonding between SC and the IgA subunits, an arrangement which follows disulfide interchange, several models for the disulfide linkage of J chain and the bonds between IgA subunits were envisaged and investigated. When sIgA was gel filtered through Sephadex G-200 in acetic acid, a single major symmetrical peak eluted at the front. This material contained SC, alpha and L chains, and all of the J chain. The greater resolution afforded by polyacrylamide gel electrophoresis in detergent confirmed that human sIgA contains no major noncovalently linked components in the 150,000-200,000 molecular weight range. In another series of experiments the Fc monomer, which is not covalently attached to SC, isolated after treatment of sIgA with IgA protease and cyanogen bromide, was investigated to learn whether alpha chain COOH-terminal octapeptides could be released by reduction. The results were negative. The available data thus favor a model in which J chain is disulfide-bonded to both IgA monomer subunits in sIgA.     

Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector

Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric-dimeric IgA (IgA(p/d)) antibody associated with secretory component (SC). SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia. This property makes SIgA suitable as potential mucosal vaccine delivery vector. It remains that production and purification of SIgA is a complex task since IgA(p/d) and SC are naturally synthesized by two different cell types. Furthermore, only IgA(p/d) are capable to associate with SC. Thus, we sought to separate IgA(p/d) and monomeric IgA (IgA(m)) antibodies secreted by hybridoma cells in CELLine bioreactors. To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system. In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors. To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgA(p/d), and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads. Biochemical characterization based on SDS-PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure. Such preparations are currently used to immunize mice to induce mucosal and systemic responses.     

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems

Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain. We present the analysis of both the N- and O-glycans on the individual peptides from this complex. Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains. The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked beta1-4 and beta1-3 to GlcNAc, fucose-linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, and alpha2-3 and alpha2-6-linked sialic acids. These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity. We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannose-binding lectin, by disrupting the SC-H chain noncovalent interactions.     

Chromatographic Separation and Purification of Secretory IgA from Human Milk

Defatted and decaseinated human milk was concentrated and was fractionated on a preparative DEAE cellulose column. Elution with various concentrations of sodium chloride in Tris-HCl buffer (pH 8.0, 0.01 M) resulted in fractions that were rich in either secretory immunoglobulin A (SIgA) (0.1 M Nad) or free secretory component (SC) (0.05 M NaCl). The fractions, which were eluted with 0.10 M NaCl from the preparative column, were further fractionated on a G-200 Sephadex column. Repeated fractionation on this column resulted in a single purified fraction, which contained very high SIgA activity and showed immunological cross-reaction with both SC and serum IgA. Additional studies indicated that this fraction was homogeneous as shown by immunoprecipitin and disc gel electrophoresis. Injection of this purified SIgA into rabbits resulted in the production of monospecific antiscil.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Peculiar secretory IgA system identified in chickens. II. Identification and distribution of free secretory component and immunoglobulins of IgA, IgM, and IgG in chicken external secretions.

A homologue of a free secretory component (SC) was identified in chicken intestinal secretion by criteria based on its antigenic relationship with intestinal secretory IgA (SIgA), molecular size, sugar content, and electrophoretic mobility, as well as its elution characteristic from ion-exchange chromatography. SC was obtained in a form free from IgA from the intestinal secretion by salting out and DEAE chromatography, followed by density ultracentrifuguation or Sephadex G-200 gel-filtration. However, the free SC revealed some antigenic deficiency when compared to bound SC of intestinal SIgA and showed a failure of binding to serum-type-polymeric IgA of biliary IgA in vitro. Several kinds of chicken external secretions were examined for detection of SC and immunoglobulin classes of IgG, IgA, and IgM. In spite of the wide distribution of immunoglobulins in the external secretions, SC antigen could be detected only in intestinal secretion. Most IgA in the secretions had a molecular structure of a tetramer of serum-type IgA, lacking in SC and having 17S to 18.5S and 600,000 to 700,000 daltons. On the other hand, IgA in the intestinal secretion showed close similarity to the mammalian SIgA, associated with SC and having 11.2S and 350,000 daltons. Presence of antibody activity in the intestinal IgA to avian reovirus was confirmed by plaque reduction tests.     

Method of preparation and evaluation of antisera for the differential determination of secretory immunoglobulin A and free secretory component in biological fluids]

One of the pressing tasks in the study of local nonsusceptibility to infectious diseases and immunochemical analysis of the external secretion is recording of the level of various forms of the secretory IgA (SIgA) and of the secretory component (SC) in various biological fluids. Indication and measurment of the concentrations of the mentioned proteins encounter serious difficulties caused by heterogeneity of their molecular forms. It was shown that the antisera to the whole molecule of SIgA and SC are of no use. On the basis of a new method of purification of free SC and technology of preparation of monospecific antisera capable of separation of SIgA and free SC there were obtained diagnostic antisera for the quantitative recording and differentiation of various forms of IgA and SC in biological fluids. A reliable measurement of the SIgA and SC concentration in some external secretion was carried out with the aid of the mentioned preparations without any complicated chromatographic experiments.     

猕猴和小鼠分泌片的分离纯化及某些生化特性

为了深入了解分泌片和ＩｇＡ在粘膜免疫系统中的作用,自猕猴和小鼠胆汁中提取和纯化分泌片,ＳＤＳ－ＰＡＧＥ结果表明猕猴和小鼠游离分泌片的分子量均约为６０ｋＤａ,但在非解聚型聚丙烯酰胺梯度凝胶电泳时分子量分别为７４ｋＤａ和６２ｋＤａ。采用ＬＫＢ－８１００等电聚焦柱测定其ＰＩ范围,猕猴分泌片为４．３－５．９,小鼠分泌为３．９－５．４。免疫双扩散证明,猕猴和小鼠胆汁中的分泌片均能与兔抗人分泌片免疫血清发生交     

SIgA分泌片分离、纯化研究

为研究SIgA分泌片（Secretory component,SC)分离,纯化及鉴定方法,以SC存在游离和结合两种形式,本研究应用凝胶过滤和盐析法直接从初乳中分离纯化游离SC,并进行鉴定。分离纯化获得蛋白溶液12.4ml,蛋白含量0.85mg/ml,免疫双扩仅与抗SC多克隆抗体（PcAb)反应,分子量约75kD,免疫印迹实验与抗SC单克隆抗体（McAb)和PcAb特异反应,表明纯化蛋白为SC。该SC的分离纯化和鉴定处于不断完善之中,其方法的改进有利于获得更纯的SC,值得粘膜免疫研究者借鉴。     

SpsA, a novel pneumococcal surface protein with specific binding to secretory Immunoglobulin A and secretory component

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10⁻⁹ M. Free secretory component (SC) also binds to S . pneumoniae , whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S . pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S . pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.     

Physicochemical and immunochemical studies on bovine IgA and glycoprotein-a

1. Bovine secretory IgA (SIgA) from colostrum (mol. wt. about 410,000) is composed of four alpha-chains (mol. wt. 61,000), four light chains (mol. wt. 23,000) and one molecule of glycoprotein-a (mol. wt. 70,000-86,000). The alpha-chains are antigenically and physicochemically distinct from the heavy chains of IgM, IgG1 and IgG2 while the light chains are identical to those occurring on other bovine immunoglobulins. Glycoprotein-a and bovine free secretory component are identical and the former name should be abolished. Much of this protein is covalently bonded to IgA. 2. The gel filtration behavior of serum IgA suggests it is a dimer. 3. The elution behavior of IgA and SIgA from ion-exchange columns and the solubility characteristic of SIgA in the presence of Zn2+ are similar to those of human and rabbit IgA. 4. The disc electrophoretic behavior of IgA and SIgA are distinct from IgM, dimeric IgG1, 7-S IgG and glycoprotein-a. Dimeric IgG1 (s20,w = 9.5) is abundant in colostrum and is similar in size to SIgA. 5. Bovine IgA shows physicochemical and immunochemical heterogeneity when studied by gel filtration, disc electrophoresis, immunoelectrophoresis and ultracentrifugational analyses. Lacrimal and nasal SIgA possess antigenic determinants absent on colostral SIgA.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.     

抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

Immunoglobulin A (IgA) polymerization sites in human immunocytes: immunoelectron microscopic study

The cytoplasmic affinity of polymeric IgA for secretory component (SC) and the expression of joining (J) chain were examined in pokeweek mitogen (PWM)-stimulated human peripheral blood lymphocytes (PBL) to determine, on the ultrastructural level, the polymerization sites of human IgA. SC-binding was found in 5.7% of transformed PBL on day 7 of culture; SC-binding was observed in a high proportion of IgA-producing cells. A low proportion of IgM-producing cells also bound to SC, while there was virtually no SC-binding by IgG-producing cells. A high proportion of IgA- and IgM-producing cells expressed intracellular J chain, while approximately half of the IgG-producing cells were positive for J chain. The number of J chain-positive cells exceeded the number of SC-binding cells among transformed PBL on day 7 of culture. Immunoelectron microscopic study of the sites of SC-binding, and of IgA and J chain expression, revealed that polymerization of human IgA and the addition of J chain occur in the perinuclear space and endoplasmic reticulum, prior to immunoglobulin secretion.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.     

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.