Isolation, Properties, and Structure of Human IgA Myeloma Globulins

Putnam, Frank W.; Low, Teresa L. K.; Liu, Victor; Huser, Hans; Ec, Raff; Wong, FS; Clamp, J. R.

doi:10.1007/978-1-4613-4550-3_20

Cited by 6 publications

(3 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Some of the results have been documented (7,9,10,17), and articles giving the complete proof of the al and a2 chain sequences are in preparation. To clarify presentation for the three proteins we summarize the results of the analysis of sequences containing glycopeptides in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The procedure involved ammonium sulfate precipitation, ion exchange chromatography, and gel filtration techniques (9,10). The purity of the IgA was tested by immunodiffusion and immunoelectrophoresis with monospecific antisera and by gel electrophoresis and ultracentrifugation (9,10). The purified IgA proteins were reduced at room temperature under N2 with 0.01 M dithiothreitol for 1 hr followed by alkylation with iodoacetic acid (10% molar excess) or ethylene imine.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Location and structural significance of the oligosaccharides in human Ig-A1 and IgA2 immunoglobulins.

Toraño¹,

Tsuzukida²,

Liu³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The location, number, and kinds of oligosaccharides in human IgAl and IgA2 immunoglobulins have been determined by amino acid sequence analysis of the a heavy chains. Both A2m allotypes of the a2 chain of IgA2 have two GlcN oligosaccharides that are absent in the al chain, but they lack GalN. The A2m(2) allotype has a fifth GlcN oligosaccharide. The a chains of IgA proteins also have subclass-specific and allotype-specific differences in amino acid sequence. Although other classes of human immunoglobulins differ in the number and kind of oligosaccharides, the sites are often homologous and are related to the immunoglobulin domain structure. Evolutionary preservation of the tripeptide acceptor sequence for GlcN probably indicates both a structural and biological role for carbohydrate.Although the five classes of human immunoglobulins differ greatly in their content of carbohydrate and in its distribution along the polypeptide chain, the carbohydrate often is in homologous positions and usually seems to lie in between the compact domains of the heavy chain or on the surface of a domain. With rare exceptions governed by the presence of a signal acceptor tripeptide sequence, the carbohydrate is present only on the heavy chain and is confined to the constant (C) region of the heavy chain. We earlier reported the location and nature of the five oligosaccharides of the At chain of human IgM (1, 2). In contrast, the oy chain of human IgG has a single oligosaccharide (3), and the E chain of human IgE is reported to have six (4). In all these cases the oligosaccharide contains glucosamine (GlcN), which is attached to asparagine by an N-glycosidic linkage. The IgAl subclass is unusual among glycoproteins in having two types of linkage to the polypeptide chain, the N-glycosidic linkage of GlcN to asparagine and an O-glycosidic linkage of galactosamine (GalN) to serine (5, 6). Recently we reported (7) the complete amino acid sequence of a human IgAl immunoglobulin (designated Bur), including the location of five GalN carbohydrates in the hinge region, two GlcN oligosaccharides in the Fc region, and an adventitious GlcN oligosaccharide in the variable (V) region of this al heavy chain. We have now determined the amino acid sequence of the C region of two a2 heavy chains representing different allotypes of the IgA2 subclass, namely, the A2m (2) MATERIALS AND METHODSHuman IgA was prepared from the serum of patients with multiple myeloma who produced large amounts of monoclonal IgA. The procedure involved ammonium sulfate precipitation, ion exchange chromatography, and gel filtration techniques (9, 10). The purity of the IgA was tested by immunodiffusion and immunoelectrophoresis with monospecific antisera and by gel electrophoresis and ultracentrifugation (9, 10). The purified IgA proteins were reduced at room temperature under N2 with 0.01 M dithiothreitol for 1 hr followed by alkylation with iodoacetic acid (10% molar excess) or ethylene imine. The heavy and light chains were separated on a Sephadex G-100 column. The...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Location and structural significance of the oligosaccharides in human Ig-A1 and IgA2 immunoglobulins.

Toraño¹,

Tsuzukida²,

Liu³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…MATERIALS AND METHODS Assay system. IgA, was isolated from human myeloma serum by the method of Putnam et al (18) and labeled with '25I (New England Nuclear Corp., Boston, Mass.) by using a modification of the method described by Marchalonis (13).…”

mentioning

confidence: 99%

Studies on gonococcus infection. XVI. Purification of Neisseria gonorrhoeae immunoglobulin A1 protease

Blake¹,

Swanson²

1978

Infect Immun

View full text Add to dashboard Cite

A protease which cleaves human immunoglobulin A1 (IgAj) has been purified from broth cultures of Neisseria gonorrhoeae. This IgAj protease is produced by pilated and nonpilated gonococci throughout their growth cycles. A combination of ammonium sulfate precipitation, column chromatography, and either isoelectric focusing or affinity chromatography was utilized to obtain an enzyme preparation that showed approximately 3,800-fold purification and exhibited two bands (65,000 and 70,000 daltons) by analytical polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate and reducing conditions. IgAj protease activity is dependent on divalent cations and is heat labile. Detection and quantitation of IgA protease activity utilized an assay in which [251I]IgA, is incubated with protease preparations and the cleavage products are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.In 1971 Mueller described a substance in culture supematants of Neisseria gonorrhoeae and several other bacterial species which, when reacted with serum proteins, caused immunoglobulin A (IgA) to migrate more negatively in immunoelectrophoresis (14). This phenomenon was explained when Plaut et al. described a protease in gonococcal culture broth which cleaves IgA1 in the hinge region to form Fab and Fc fragments (16).A role of this enzyme in gonococcal infections or in modifying the host immune response has not been established. Antibodies to gonococcal antigens can be found in sera of patients who have had gonorrhea (1-3,8), but because uncomplicated, acute gonorrhea is largely confined to mucosal surfaces, the local immune response is of greater theoretical importance in this infection. O'Reilly and co-workers demonstrated the presence of secretory immunoglobulins specific for gonococci in exudates from both male and female patients with gonorrheal infections (7, 8).These results plus our findings that IgAj protease can be detected in vaginal washings of women with gonorrhea (M. S. Blake et al., J. Infect. Dis., in press) suggest that there could be interactions between secretory IgA (sIgA) and gonococcal protease in the course of gonorrheal infections. This paper describes methods to purify IgAj protease from culture supernatants of N. gonorrhoeae. t Present address: The Rockefeller University, New York, NY 10021. (This manuscript was submitted to the University of Utah by M.S.B. in partial fulfillment of the requirements for the Ph.D. degree in experimental pathology.) MATERIALS AND METHODS Assay system. IgA, was isolated from human myeloma serum by the method of Putnam et al. ( 18) and labeled with '25I (New England Nuclear Corp., Boston, Mass.) by using a modification of the method described by Marchalonis (13). This method was modified only by using lactoperoxidase (Sigma Chemical Co., St. Louis, Mo.) which had been coupled to Sepharose 4B (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) (4). Unbound '25I was removed by filtering the solution over a Sephadex G 25 column (0.9 by 15 cm) equilibrated with 0.05...

show abstract

Complete amino acid sequence of the alpha 2 heavy chain of a human IgA2 immunoglobulin of the A2m (2) allotype.

Toraño¹,

Putnam²

1978

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The complete amino acid sequence of the a2 heavy chain of a human IgA2 immunoglobulin of the A2m(2) allotype has been determined and is compared to the sequence of the al chain of the human IgAl subclass. The characteristic differences between the al and a2 chains are greatest in the hinge region and in the location and number of the oligosaccharides. Apart from the duplication in the hinge region of al and the deletion in a2, there are 23 amino acid exchanges in the constant (C) regions of the two chains. Accepted mutations are related to the surface accessibility of the residues and the proximity of carbohydrate. The results indicate that human IgA and IgG subclasses arose late in evolution and reflect similar mutational pressures.IgA, the characteristic antibody of secretory fluids and the second most abundant of the five classes of human immunoglobulins, normally exists as two well-defined subclasses, IgAl and IgA2, that can be differentiated both chemically and antigenically (1, 2). By serological methods, a genetically controlled polymorphism (allotypy) has been identified in the IgA2 subclass. The two allotypes of IgA2 have been given various confusing designations but the recommended notation is now A2m(1) and A2m(2). These terms correspond to the notation Am2(+) and Am2(-) used earlier by us (3) and by others. Recently, an isoallotype of human IgA proteins, designated nA2m(2), was identified serologically by van Loghem et al. (4). Our objectives have been to determine the primary structure of human IgAl and IgA2 proteins, including the complete amino acid sequence of their heavy chains (al and a2, respectively), to establish the location and kinds of oligosaccharides, to identify the genetically determined differences in structure of the allotypes, and to relate these to the biological function, three-dimensional structure, and evolution of the IgA class of molecules. We have previously reported the complete covalent structure of an IgAl myeloma protein (designated Bur) (5), the comparison of the Fc structure of human IgA, IgG, IgM, and IgE (6), and the location and nature of the oligosaccharides in human al and a2 heavy chains as well as some amino acid substitutions characteristic of the subclasses and allotype of a chains (7).We report here the complete amino acid sequence of the a2 heavy chain of a human A2m(2) myeloma protein (designated But). The frequency of this allotype is highest in Africa, is less in Australia and Asia, and is lowest in Europe (2). Subclasses and allotypes are also known for human IgG, but only partial sequences of the -y chains of the four human subclasses of IgG have been published; they have been summarized by Beale and Feinstein (8). Overall, the al chain and the a2 chain of the A2m(2) allotype are remarkably similar; the major differences are: (i) the partial duplication in the al hinge and the deletion in the a2 hinge already described (7, 9); (ii) the number and location of the oligosaccharides (7); and (iii) 23 amino acid substitutions outside the hinge regio...

show abstract

Isolation, Properties, and Structure of Human IgA Myeloma Globulins

Cited by 6 publications

References 14 publications

Location and structural significance of the oligosaccharides in human Ig-A1 and IgA2 immunoglobulins.

Location and structural significance of the oligosaccharides in human Ig-A1 and IgA2 immunoglobulins.

Studies on gonococcus infection. XVI. Purification of Neisseria gonorrhoeae immunoglobulin A1 protease

Complete amino acid sequence of the alpha 2 heavy chain of a human IgA2 immunoglobulin of the A2m (2) allotype.

Contact Info

Product

Resources

About