Human IgD Research Articles

A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD

The α- D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95–99% pure as determined by SDS-PAGE and represents 90–95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.

The gene sequence and some properties of protein H. A novel IgG-binding protein.

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.

Class switch from μ to δ is mediated by homologous recombination between σμ and σμ sequences in human immunoglobulin gene loci

Class switch of immunoglobulin from mu to gamma occurs by recombination between two repetitive switch sequences: S mu and S gamma. However, there are no such sequences in the mu-delta introns of human and mouse genomes. Although the frequency of IgD-secreting cells is extremely low in mouse about 1% of patients with myeloma produce IgD in human. In a previous report (Nucleic Acids Res. 1988. 16: 9497) we reported that a 442-bp DNA sequence located in the JH-mu intron (defined as sigma mu) was inserted into the mu-delta intron (defined as sigma mu) in human genome. There is no such insertion in mouse. We analyzed Ig H chain gene loci of two human IgD myelomas: one was analyzed by cloning and sequencing and the other by Southern hybridization. We found that recombination had occurred between these two homologous DNA sequences, resulting in loss of the DNA segment from sigma mu to sigma mu. On the other hand, in a Burkitt lymphoma, Daudi, the DNA fragment from sigma mu to sigma mu was duplicated. These results suggest that homologous recombination between sigma mu and sigma mu sequences mediates class switch from mu to delta in human and that it occurs via unequal crossing-over between sister chromatids or daughter chromosomes.

Quantitative assay of a human monoclonal IgM antibody (HA-1A) in human serum

A rapid, specific and sensitive radiometric assay was developed capable of quantitating serum levels of HA-1A, a human IgM monoclonal antibody to endotoxin. ‘Private’ anti-idiotypic murine monoclonal antibodies were produced and utilized in the assay to avoid cross-reactivity with normal human IgG, IgM, IgA, IgE or IgD. The presence of E. coli or gram-negative lipopolysaccharide in the sera did not affect the ability of the assay to detect HA-1A. The sensitivity of the assay was calculated to be 25 ng/ml with an interassay coefficient of variation of less than 10%. In one patient given 100 mg of HA-1A, peak serum concentration was 101.5% of the predicted value with a mean plasma half life of 24.5 h. This assay will be useful in establishing the pharmacokinetics of HA-1A and in monitoring serum levels during phase II and phase III clinical trials.

The human immunoglobulin Cμ–Cδlocus: complete nucleotide sequence and structural analysis

The entire nucleotide sequence (approximately 20 kbp) spanning the human immunoglobulin IgM (mu) and IgD (delta) heavy chain constant region genes has been determined from DNA of mu-delta producing chronic lymphocytic leukemic B cells. As in the murine IgM + IgD double-producing B cells, no rearrangement has occurred in the C mu-C delta region in the leukemic cells. The C mu locus is highly conserved between mouse and human with the exception of the nucleotide sequence between the C mu 4 and mu M1 exons, which has diverged dramatically. The intergenic sequence between human C mu and C delta is three times larger than the analogous region in the mouse and contains notable features absent from the mouse, including a 443 bp segment that is 96% identical to a 442 bp sequence that occurs just 3' to the heavy chain enhancer, a 366 bp sequence that is directly repeated with 76% homology, and 12 tandem copies of a 35 bp sequence. The human C delta gene contains two additional exons relative to mouse C delta, but shares with the mouse the unique distal location of both secreted and membrane coding segments. Several polymorphisms in the human population have been identified in the intergenic region and in C delta but not in C mu.

Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

In vitro studies on human IgD : III. Immunologic features of individuals with high sera IgD and spontaneous IgD biosynthesis

The immunologic characteristics of normal persons with high sera IgD values were analyzed. Elevated sera IgD appeared to be the consequence of increased biosynthesis as reflected by an increased number of IgD-Ig-containing cells, an elevated sera IgD, λ κ ratio, and increased spontaneous IgD secretion in vitro. These same findings have been previously linked to increased IgD production (S. D. Litwin and B. D. Zehr, Eur. J. Immunol. 17, 483, 491, 1987). Elevated sera IgD proved relatively stable over 21 months, in five selected individuals, favoring a genetic vs acquired explanation. The failure of sera IgG, IgA, and IgM to positively correlate with IgD weighed against polyclonal Ig synthesis. However, high IgD was inversely correlated to IgM class values and a high sera IgD subset of the population (>31 μg/ml) had lower levels of certain IgM antibodies. Analysis of spontaneous IgD secretion, an event frequently encountered in high sera IgD persons, disclosed 2 29 cultures with rising supernatant IgD implying in vitro induction. The results emphasize a role for active IgD biosynthesis in the immune responses of certain individuals.

Human IgD and IgA1 compete for D-galactose-related binding sites on the lectin jacalin.

Lectin selectivity for human Ig classes is based on carbohydrate differences. Earlier reports that the lectin jacalin precipitated human IgA were confirmed and supplemented by the current study, which demonstrates that jacalin also binds human IgD as evaluated by micro-ELISA and SDS-PAGE. Experimental findings indicated that: (i) Monoclonal and polyclonal (sera) IgD, IgA1, but not IgA2, IgM, or IgG1-4 reacted with jacalin. (ii) Six tested monoclonal IgD proteins each bound approximately equally to jacalin when antigenicity rather than protein concentration was measured: the results weigh against the presence of jacalin-detectable IgD subclasses or genetic variants. (iii) IgD and IgA1 both associated maximally in 4-8 h at 4 degrees C. There was no dissociation at 4 degrees C but limited dissociation occurred at 37 degrees C after 24 h. (iv) Both IgD and IgA1 were eluted from jacalin by galactose-related sugars. (v) IgD and IgA1 bind competitively to jacalin. The results suggested that jacalin reacts with O-linked oligosaccharide N-acetyl-galactosamine (GalN) residues found on the hinge region of both IgD and IgA1. Jacalin also interacted with one major and several minor unidentified sera proteins. The findings offer an approach to the isolation of serum polyclonal IgD and to the characterization of the unusual carbohydrates of the human delta heavy chain with respect to their function.

Characterization of jacalin, the human IgA and IgD binding lectin from jackfruit

The lectin jacalin from the jackfruit Artocarpus heterophyllus reacts by precipitation and western blotting with human IgA1 and IgD but not with IgA2 (nor IgG and IgM). However, it weakly binds IgA2 as shown by affinity chromatography and competitive ELISA. Predominantly reactive carbohydrates are d-galactose and N-acetyl d-galactosamine. Jacalin has an apparent M r of about 54,000 and is probably made up of three non-glycosylated and one glycosylated non-covalently linked subunits. Its electrophoretic properties and amino acid and carbohydrate composition are indicated.

In vitro studies on human IgD. I. Sources and characteristics of "externalized" IgD in tonsil lymphocyte cultures.

As part of a broader analysis into the function of IgD, and especially into the role of human IgD-secreting cells, fresh human tonsil lymphocyte cultures were analyzed. The goals were to define the origins of "externalized" supernatant IgD and to examine its relationship to spontaneous Ig secretion of IgG, IgM and IgA. Assayable IgD was found by 24 h in cultures without added mitogens. "Externalized" IgD comprised a small fraction (1.6-1.8%) of supernatant Ig when compared to the fraction of IgD-containing cells (congruent to 10%): IgD appears to disappear rapidly in vitro. Initial experiments employing irradiation and chemical inhibitors of protein synthesis suggested that "externalized" IgD was produced de novo during culture rather than representing preformed or cytophilic Ig. Most significantly, tonsil cultures with higher IgD values (greater than 20 ng IgD/10(6) cells, day 7) showed convincing evidence that secreted IgD was a major source for "externalized" IgD. This evidence included the amount of "externalized" IgD depended on viable culture conditions; radioincorporated 35S appeared as a visible IgD band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels; "higher" IgD cultures showed increased numbers of IgD-Ig-containing plasma cells, and IgD, lambda in excess of IgD, kappa; both events are associated with IgD secretion. In cultures producing high amounts of IgD, IgD secretion appeared to be spontaneous rather than induced and was independent of the polyclonal spontaneous Ig secretion of other Ig isotypes. Low titers of inhibitable IgD isotype anti-phosphorylcholine antibodies but not IgD isotype anti-polyribotol phosphate antibodies were present in tonsil supernatants: both were present in matched sera. Other laboratories have directed attention to the striking and possibly selective participation of human tonsil lymphocytes in IgD synthesis using other approaches. Present results supplement and expand earlier data and support the practical value of analysis of short-term cultured tonsil lymphocytes. Differentiation to IgD-secreting cells is suggested to be an active, underestimated and putatively an important part of the immune response in the human tonsil.

Interaction between human IgD and ricinus agglutinin.

The results show that monoclonal IgD reacts specifically with ricinus agglutinin (molecular weight 120,000) (RcAI). The reaction between IgD and RcAI was inhibited by galactose and lactose but not by glucose, indicating that the interaction is mediated by galactose containing carbohydrate units located in or near the sigma-hinge region. Affinity chromatography on RcAI-Sepharose 4B was successfully used to isolate monoclonal IgD from the IgD myeloma plasma.

Anti-IgE autoantibody in patients with atopic dermatitis.

The anti-IgE autoantibody in the IgG class was detected in 39/45 (86.7%) patients with atopic dermatitis by using a newly established solid-phase enzyme immunoassay. The epsilon-chain specificity of the anti-IgE autoantibody was confirmed by competitive inhibition by using human IgG, IgA, IgM, IgD, IgE, and heat-denatured IgE protein. Significant correlations were observed between the levels of the anti-IgE autoantibody and the serum IgE. Gel filtration studies indicated that the anti-IgE autoantibody in sera from atopic dermatitis was mainly present in the form of an immune complex with self IgE. The role of the IgE-anti-IgE immune complex and the role of the anti-IgE autoantibody in the modulation of the IgE-mediated immune system in atopic dermatitis are discussed.

Studies on human low serum IgD phenotype and Gm markers.

The human "low serum IgD phenotype" was studied by simultaneous Gm typing and IgD immunoassay of several populations. An association between Gm (f+b+) haplotype and low human IgD was confirmed and extended to the "low serum IgD phenotype"--as defined from population distribution and genetic studies by Dunnette et al. 1978. Further, it was shown that Black American sera determined by Gm haplotype, had a similar percentage of "low serum IgD phenotype" samples (16%) although they lacked the "associated" Gm(f+b+) haplotype of White American samples. Sardinian sera showed a low incidence of the "low serum IgD phenotype" which was not correlated with Gm haplotype distribution. Familial aggregation of the "low serum IgD phenotype" was observed. No association was found between "low serum IgD phenotype" and serum IgE values. Age related abiotrophy of IgD could not be attributed to selective survival of "low serum IgD phenotype" persons.

An avidin-biotin ELISA for the measurement of serum and secretory IgD

This paper described an improved microtiter solid-phase enzyme immunoassay for the determination of serum and secretory IgD. Use of the interaction between biotinylated anti-human IgD and horseradish peroxidase(HRP)-avidin conjugate permits quantitation of human IgD in the range of 1–64 ng/ml. IgD was detected in all samples of serum, saliva and nasal secretions of 28 normal adults. In only one subject both serum and secretory IgD were undetectable. The mean concentration of serum IgD determined by this assay is similar to that reported by other authors using radioimmunoassay. The assay described is not only rapid and inexpensive but at least as sensitive as the radioimmunoassays usually employed for quantitation of IgD.

The sequence of the human immunoglobulin μ-δ intron reveals possible vestigial switch segments

We present the full sequence of an insert of a lambda phage clone which contains a segment of human DNA stretching from the secreted mu(mu s) constant region gene through to the beginning of the constant region gene and including the membrane mu(mu m) segments. The segment of 8.6kb extending from mu s to the first constant domain of delta(C delta 1) has been completely sequenced and reveals little conservation in comparison to the corresponding mouse sequence. The outstanding feature of the mu s-mu m intron is the occurrence of a potential Z-DNA forming region situated at 285bp downstream of the mu s poly A addition signal. A similar DNA stretch exists in mouse and may represent a site for transcriptional control of mu gene expression. The mu m-C delta 1 intron is much longer (6Kb) than the corresponding mouse intron and includes a series of different repeats, which start at 430bp downstream of the mu m poly A addition site and continue for 3.5Kb, ending about 1.5Kb from the beginning of C delta 1. This series of repeats may be a vestigial switch sequence used in the production of the secreting cells which are the progenitors of the rare human IgD myelomas.

Cross-reactivity of human and a putative pig IgD. Pig IgD-like molecules as serum and lymphocyte components.

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, and susceptibility of the heavy chains, light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.

The appearance of an IgD-like molecule on pig lymphocytes during ontogeny.

An IgD-like molecule was found on the surface of pig fetal lymphocytes using guinea pig anti-human IgD antisera and indirect immunofluorescence. The binding of antiIgD antisera could be specifically inhibited with human IgD. Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen). There is evidence for their endogenous origin. The number of lymphocytes bearing IgD-like molecules rapidly increased during ontogeny.

Structures of the oligosaccharides present at the three asparagine-linked glycosylation sites of human IgD.

The complete amino acid sequence of the human myeloma IgD:WAH has been determined and the sites of asparagine glycosylation identified as residues 354, 445, and 496 (Takahashi, N., Tetaert, D., Debuiere, B., Lin, L.-C., and Putnam, F. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2850-2854). We have determined the structures of the oligosaccharides at each of these positions. Asn 354 bears oligosaccharides exclusively of the high mannose type containing from 5 to 9 mannose residues. Twenty per cent of the oligosaccharides at this site contain 1 glucose residue at the terminus of the branch emanating from the alpha 1 leads to 3-linked core mannose which is believed to reflect incomplete processing of the triglucosyl-high mannose oligosaccharide intermediate following transfer from dolichol to nascent peptide. Asn 445 and Asn 496 bear exclusively dibranched complex oligosaccharide structures; 30-40% of these molecules contain a bisecting GlcNAc-linked beta 1 leads to 4 to the innermost core mannose residue. At Asn 445, 40% of both the bisected and nonbisected oligosaccharides contain 1 residue of fucose on the Asn-linked GlcNAc and 50% bear a single N-acetylneuraminic acid residue. The oligosaccharides at Asn 496 are devoid of sialic acid and fucose. Thus, IgD:WAH is notable for the presence of virtually unprocessed oligosaccharide structures (glucosylated high mannose) on the same peptide backbone as extensively processed complex type molecules. The finding that each of the 3 Asn glycosylation sites of IgD:WAH bears either exclusively a complex or a high mannose type oligosaccharide indicates that there is considerable specificity in the glycosylation process. These oligosaccharides, nonetheless, display extensive microheterogeneity at each location.

Structures of the O-glycosidically linked oligosaccharides of human IgD.

In the previous communication (Mellis, S. J., and Baenziger, J. U. (1983) J. Biol. Chem. 258, 11546-11556), the structures of the oligosaccharides present at the 3 asparagine glycosylation sites of a human IgD myeloma protein were defined. In this communication, we present the structures of the O-glycosidically linked oligosaccharides located in the hinge region of IgD:WAH. Three or four threonine residues and one serine residue in the region bear O-glycosidically linked oligosaccharides. Approximately 50% of these molecules have the structure Gal beta 1 leads to 3 GalNAc which is identical with the structure of the predominant oligosaccharide in the hinge region of human IgA1 myeloma proteins (Baenziger, J. U., and Kornfeld, S. (1974) J. Biol. Chem. 249, 7270-7281). The remainder of the oligosaccharides contain 1 or 2 residues of N-acetylneuraminic acid and have the structures NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc (30%), Gal beta 1 leads to (NeuAc alpha 2 leads to 6)GalNAc (12%), and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc (8%). The sialylated molecules have not been encountered previously on other human immunoglobulin heavy chains. These structures, however, have been described on a number of secreted and membrane glycoproteins. Examination of oligosaccharides isolated from different subregions of the IgD hinge indicated that a specific distribution of the sialylated structures among the glycosylated amino acids of the hinge region is not likely.

Two-dimensional high-performance liquid chromatography and chemical modification in the strategy of sequence analysis : Complete amino acid sequence of the lambda light chain of human immunoglobulin D

Rapid high-performance liquid chromatographic procedure forms part of the strategy for determining the amino acid sequence of proteins. It was applied to the light chain of an immunoglobulin D, and the result, together with previous data on the sequence of the heavy chain, established the complete covalent structure of a human IgD immunoglobulin. We aimed for rapid purification of both small and large peptides. The two-step chromatography system (“two-dimensional chromatography”) consists of a combination of macroreticular cation-exchange chromatography as the first dimension and reversed-phase chromatography as the second. This procedure was used for the systematic separation of the tryptic peptides of the light chain. In order to obtain overlapping of the tryptic peptides, chemical modifications of the light chain and reversed-phase chromatography were found to be the most reliable path toward the sequence analysis and greatly accelerated the determination of the light chain.