Protein Rou. A human IgA hybrid

Protein Rou is a human IgA2 myeloma protein that carries the isoallotype marker n A2m(2). Partial amino acid sequence of its H chain (alpha) shows that the hinge region and the CH2 domain are homologous to alpha 2-chain and the CH1 and the CH3 domains homologous to alpha 1. Moreover, the CH1 domain contains the H-L disulfide bond identical to alpha 1. It is concluded that Rou H chain is a hybrid molecule caused by a recombination between alpha 1 and alpha 2 genes. The recombination event occurred between alpha 1-exon 1 and alpha 2-exon hinge and corresponds to position 222-223 of the alpha-chain.     

O-linked glycosylation in maize-expressed human IgA1

O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis. Approximately 90% of hIgA1 was modified in this way. An average molar ratio of six Ara units per molecule of hIgA1 was revealed. Substantial sequence similarity was identified between the HC hinge region of hIgA1 and regions of maize extensin-family of hydroxyproline-rich glycoproteins (HRGP). We propose that because of this sequence similarity, the HC hinge region of maize-expressed hIgA1 can become a substrate for posttranslational conversion of Pro to Hyp by maize prolyl-hydroxylase(s) with the subsequent arabinosylation of the Hyp residues by Hyp-glycosyltransferase(s) in the Golgi apparatus in maize endosperm tissue. The observation of up to six Pro/Hyp hydroxylations combined with extensive arabinosylation in the hIgA1 HC hinge region is well in agreement with the Pro/Hyp hydroxylation model and the Hyp contiguity hypothesis suggested earlier in literature for plant HRGP. For the first time, the extensin-like Hyp/Pro conversion and O-linked arabinosylation are described for a recombinant therapeutic protein expressed in transgenic plants. Our findings are of significance to the field of plant biotechnology and biopharmaceutical industry-developing transgenic plants as a platform for the production of recombinant therapeutic proteins.     

An abnormal human IgA1 half-molecule.

An IgA1 half-molecule, which is composed of a deleted alpha1 chain linked with a disulfide bond to an intact kappa chain, was detected in a patient (Cha). The molecular weights of the paraprotein and the isolated alpha1 chain were estimated to be 75 000 and 53 000, respectively. Identification of tyrosine as the C-terminal amino acid and the presence of idiotypic determinants in the abnormal alpha1 chain indicated that the molecule would have an intact N-terminal variable region and a C-terminal region. Furthermore, no cleavage of the abnormal protein into Fab and Fc by proteolytic enzyme isolated from Neisseria gonorrhoeae suggested the absence of a "hinge" region in the abnormal alpha1 chain.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

Study of the relationship between sticky human serum IgA1 and its O-glycan glycoform.

The high-affinity IgA1 toward jacalin was mostly composed of aggregated IgA1 and abundantly contained the asialo disaccharide, Galbeta1,3GalNAc, in the O-linked oligosaccharide in the hinge region [Journal of Biochemistry 120, 92-97 (1996)]. Meanwhile, the removal of sialic acid from IgA1 accelerated the aggregation of the IgA1 molecule [J. Am. Soc. Nephrol. 9, 2048-2054 (1998)]. In order to examine the nature of such a sticky IgA1, affinity chromatography using asialo-IgA1 (deSIgA1)-Sepharose was carried out. Seventeen percent of normal human serum IgA1, 27% of asialo-IgA1 (IgA1-S), and 48% of asialo-, agalacto-IgA1 (IgA1-SG) were bound to the column. Removal of the N-acetylgalactosamine residue from IgA1-SG resulted in a decreasing affinity toward deSIgA1-Sepharose. Thus, the binding ability toward the column was the highest for the IgA1-SG among the deglycosylated IgA1s. On the other hand, heat treatment of IgA1 accelerated the aggregation but decreased its binding ability toward the column. Such heat denaturation probably destroys the structure of the binding site. Since the enzymatic removal of the N-glycan sugar chains did not induce the aggregation and exhibited no effect on the binding, the incomplete O-linked sugar chain on the hinge portion should be directly related to the sticky characteristics of the IgA1 molecule. The binding was non-covalent and not strong because the asialo-, agalacto-hinge glycopeptide was eluted slightly slower than the native one from the column and the bound IgA1 was dissociated in the presence of 1 M NaCl.     

Conformational preferences of sequential fragments of the hinge region of human IgA1 immunoglobulin molecule: II

The mean solution conformation of tetrapeptide fragments of the hinge region of human IgA1 molecule was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences were found. Tetrapeptides with the Thr-Pro-Ser-Pro sequence were found to show a clear preference for the beta-turn conformation. Conformational equilibria of these peptides are only slightly affected by acetylation or pH changes. In the case of Pro-Thr-Pro-Ser tetrapeptides conformational equilibria are dominated by unordered forms.     

Aberrant glycosylation of IgA from patients with IgA nephropathy

Despite the prominent role of IgA, particularly IgA₁, in the pathogenesis of IgA nephropathy (IgAN), the precise role of this molecule in the process remains unclear. Four biotin-conjugated lectins in sandwich-type enzyme-linked immunosorbent assays were devised to determine the glycosylation profiles of total IgA and its subclasses. We took advantage of differential binding properties of these lectins to sugar residues to dissect the oligosaccharide chainsO-linked to the hinge and thoseN-linked to the Fc region of total IgA and IgA subclasses in 47 patients with IgAN and an equal number of controls. The proportion of sialylated IgA₁ was higher in patients compared with controls (p<0.02), whereas IgA₂ in patients appeared less well sialylated. A reduction of galactose in pathological IgA as detected by RCA-I became significant after treatment of the molecule with neuraminidase (p<0.01). Defective galactosylation was also observed for patient IgA₁ when it was probed with ECL, a lectin that has a specificity for Gal 1,4N-acetylglucosamine groupings onN-linked oligosaccharides. The RCA and ECL results, therefore, suggest that increased sialylation on the IgA₁ is onO-linked oligosaccharides in the hinge region. This was partly confirmed by a small increase in the binding of PNA to IgA₁ from the patient group. This lectin binds preferentially to Gal 1,3N-acetylgalactosamine groups that are found onO-linked oligosaccharides.     

The specificity of the IgA receptor purified from human neutrophils.

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.     

Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences

In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with ¹⁵N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes.     

Transforming growth factor-beta directs IgA switching in human B cells.

Transforming growth factor (TGF)-beta added to cultures of highly purified human splenic B cells induced high levels of IgA synthesis in the presence of PWM and activated cloned CD4+ T cells. TGF-beta had no effect on IgM or IgG production. The induction of IgA synthesis by TGF-beta reflected IgA switching, because a strong induction of IgA production was also observed, when sIgA- B cells were cocultured with cloned activated CD4+ T cells in the presence of pokeweed mitogen. Resting CD4+ T cell clones or activated CD8+, TCR-gamma delta + CD4-,CD8- T cell clones failed to provide the co-stimulatory signal that in addition to TGF-beta and pokeweed mitogen was required for induction of IgA switching and IgA synthesis. mAb against CD4 or class II MHC molecules inhibited TGF-beta induced IgA synthesis, indicating that CD4-class II MHC interactions are required for productive T-B cell contacts resulting in IgA production. In contrast, anti-LFA-1, anti-CD2, and anti-class I MHC mAb were ineffective. TGF-beta failed to induce IgA synthesis by sIgA+ B cells under these culture conditions. Interestingly, induction of IgA production by sIgA- B cells required neutralization of TGF-beta activity by addition of the anti-TGF-beta mAb 1D11.1G 24 h after onset of the cultures. IgA production was prevented when the anti-TGF-beta mAb was added at the start of the cultures, indicating the specificity of the reaction. IgA synthesis was completely suppressed when TGF-beta was present during the total culture period of 11 days. These findings indicate that TGF-beta can act as a specific switch factor for IgA, provided it is only present at early stages of the cultures.     

Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1

Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure.     

IgA1 protease

IgA1 proteases are proteolytic enzymes that cleave specific peptide bonds in the human immunoglobulin A1 (IgA1) hinge region sequence. Several species of pathogenic bacteria secrete IgA1 proteases at mucosal sites of infection to destroy the structure and function of human IgA1 thereby eliminating an important aspect of host defence. IgA1 proteases are known as autotransporter proteins as their gene structure encodes the information to direct their own secretion out of the bacterial cell. The iga gene structure is also thought to contribute to the antigenic heterogeneity demonstrated by the IgA1 proteases during infections and the cleavage specificity of the IgA1 proteases for human IgA1. The IgA1 proteases have therefore been implicated as important virulence factors that contribute to bacterial infection and colonisation. The development of strategies to inactivate these IgA1 proteases has become the subject of recent research, as this has the potential to reduce bacterial colonisation at mucosal surfaces.     

Fc alpha RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.

The FcR for IgA CD89/FcalphaRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.     

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn‐binding motif (1604‐1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N‐terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C‐terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.     

Conformational preferences of the sequential fragments of the hinge region of the human IgA1 immunoglobulin molecule

The mean solution conformation of tetrapeptide fragments spanning the hinge region of human IgA1 was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences of IgA1 were found. In a series of tetrapeptides having the Thr-Pro-Pro-Thr sequence, the Pro-Pro fragment was ordered to the structure of a type II polyproline helix, but with unordered forms prevailing in the equilibria. In the case of the Pro-Pro-Thr-Pro sequence, a distinct preference for the beta-turn conformation was found. Acetylation of this tetrapeptide shifts the equilibrium towards unordered forms containing some elements of the type II polyproline helix. The peptide Thr-Pro-Ser-Pro exists predominantly in the beta-turn conformation whereas Pro-Ser-Pro-Ser-NH2 has, for the most part an unordered conformation.     

In vitro regulation of IgA subclass synthesis. II. The source of IgA2 plasma cells

In vitro regulation of IgA subclass synthesis was investigated in pokeweed mitogen (PWM)-stimulated cultures of peripheral blood lymphocytes. In past experiments we have demonstrated that 50% of the IgA plasma cells derived from PWM-stimulated cultures are positive for IgA1 and 50% are positive for IgA2. This observation is surprising because approximately 80% of the IgA B cells in the peripheral circulation bear IgA1 and 20% bear IgA2. To determine if the shift toward IgA2 predominance in PWM-stimulated cultures might be due to an enriched source for IgA2 plasma cells from a precursor pool of immature B cells, we used panning techniques to separate immature precursors that express surface IgM (sIgM+) from mature precursors that no longer express IgM (sIgM-). These separated B cells were cultured with equal numbers of T cells and PWM for 7 days. In all 10 experiments there was an enrichment for IgA2 in the sIgM+ cultures; 55 +/- 9.6% of the total IgA plasma cells were positive for IgA2 in the sIgM+ cultures vs 38 +/- 6.3% in the sIgM- cultures (p less than 0.001). These results indicate that both sIgM+ and sIgM- cells can give rise to IgA plasma cells in PWM-stimulated cultures and that there is an enrichment for IgA2 precursors in the sIgM+ population. Other possible regulatory mechanisms were also investigated. To determine if there was isotype switching from IgA1 to IgA2, monoclonal anti-IgA1 antibodies were added to PWM cultures. These antibodies resulted in a mean suppression of IgA1 plasma cell production of 82% with a concomitant 45% suppression of total IgA but only 4.6% suppression of IgA2. These results make it unlikely that IgA2 plasma cells in PWM-stimulated cultures are derived from cells that initially produced IgA1. To investigate the possibility that one IgA subclass might be more T cell dependent than the other, T and B cells were separated and B cells were reconstituted with T cells in ratios that varied from 1:10 to 10:1 or with irradiated T cells. These procedures did not alter the proportion of IgA plasma cells positive for IgA1 or IgA2, indicating that the two subclasses do not differ in their response to T cell signals in PWM-stimulated cultures.     

Structural studies of human IgA1 and IgA2 immunoglobulins labeled with two different spin labels

The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure.     

Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases

The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.     

Mapping the interaction between murine IgA and murine secretory component carrying epitope substitutions reveals a role of domains II and III in covalent binding to IgA.

We have identified sites for epitope insertion in the murine secretory component (SC) by replacing individual surface-exposed loops in domains I, II, and III with the FLAG sequence (Crottet, P., Peitsch, M. C., Servis, C., and Corthésy, B. (1999) J. Biol. Chem. 274, 31445-31455). We had previously shown that epitope-carrying SC reassociated with dimeric IgA (IgA(d)) can serve as a mucosal delivery vehicle. When analyzing the capacity of SC mutants to associate with IgA(d), we found that all domain II and III mutants bound specifically with immobilized IgA(d), and their affinity for IgA(d) was comparable to that of the wild type protein (IC(50) approximately 1 nM). We conclude that domains II and III in SC are permissive to local mutation and represent convenient sites to antigenize the SC molecule. No mutant bound to monomeric IgA. SC mutants exposing the FLAG at their surface maintained this property once bound to IgA(d), thereby defining regions not required for high affinity binding to IgA(d). Association of IgA(d) with SC mutants carrying a buried FLAG did not expose de novo the epitope, consistent with limited, local changes in the SC structure upon binding. Only wild type and two mutant SCs bound covalently to IgA(d), thus implicating domains II and III in the correct positioning of the reactive cysteine in SC. This establishes that the integrity of murine SC domains II and III is not essential to preserve specific IgA(d) binding but is necessary for covalency to take place. Finally, SC mutants existing in the monomeric and dimeric forms exhibited the same IgA(d) binding capacity as monomeric wild type SC known to bind with a 1:1 stoichiometry.     

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.