Structure and Role of the Five Glycopeptides of Human IgM Immunoglobulins

Shimizu, Akira; Putnam, Frank W.; Paul, C.; Clamp, John R.; Johnson, I. R.

doi:10.1038/newbio231073a0

Cited by 80 publications

(34 citation statements)

References 25 publications

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“…Additionally one joining chain (JC) is present to form a pentameric molecule. On each HC, five glycosylation sites are present (Shimizu et al 1971) which enable pentameric IgM molecules to become heavily glycosylated. Secreted IgM consists predominantly of pentamers of the size of *950 kDa.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulumstress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

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Section: Introductionmentioning

confidence: 99%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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“…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).…”

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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.

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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...

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“…The high-mannose oligosaccharides are circled and complex type oligosaccharides are boxed. Two oligosaccharides are enclosed in broken lines to denote that the assignment of complex type was made from homology with the human ,.t oligosaccharides (26). The linkages of three of the intrachain bridges are indicated by broken lines because they have been assigned only by assuming homology with the human A sequence Ou (11).…”

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confidence: 99%

Amino acid sequence of a mouse immunoglobulin mu chain.

Kehry¹,

Sibley²,

Fuhrman³

et al. 1979

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

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The complete amino acid sequence of the mouse ,g chain from the BALB/c myeloma tumor MOPC 104E is reported. The CA region contains four consecutive homology regions of approximately 110 residues and a COOH-terminal region of 19 residues. A comparison of this p chain from mouse with a complete ;& sequence from human (Ou) and a partial A chain sequence from dog (Moo) reveals a striking gradient of increasing homology from the NH2-terminal to the COOH-terminal portion of these It chains, with the former being the least and the latter the most highly conserved. heavy chains held together by a third type of polypeptide designated the J chain (9). It is not known whether the membrane-bound and secreted ,u chains have the same polypeptide structure. Finally, after the IgM molecule binds antigen, the C,4 domain activates the effector pathways of the complement system (10).In this paper we report the amino acid sequence of the secreted ,. chain derived from the mouse myeloma tumor MOPC 104E. The mouse ti chain is compared with a human ,u chain whose sequence has been completely determined (11) and a dog ,q chain whose sequence has been partially determined (12, 13). As previously observed when the human ,u sequence and the dog At partial sequence were compared, there is a striking gradient of sequence homology, with the COOH-terminal homology regions significantly more conserved than the NH2-terminal homology regions (12). Several structural features of the ,u chain are highly conserved, including the placement of disulfide bridges and the locations of tryptophan residues and carbohydrate moieties in the constant region. t To whom reprint requests should be addressed.

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Structure and Role of the Five Glycopeptides of Human IgM Immunoglobulins

Cited by 80 publications

References 25 publications

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

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

Amino acid sequence of a mouse immunoglobulin mu chain.

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