Biomedical subjects

M J Shulman

Publications and source records attributed to M J Shulman.

At least 55 records · Page 3Linked to original sources

PubMed1989

Mutations of the mouse mu H chain which prevent polymer assembly.

Earlier work has shown that truncated mu-chains lacking the carboxy-terminal C mu 4-tail region are secreted as monomeric rather than polymeric IgM and that the monomer phenotype is not due to the lack of a disulfide bond at Cys-575 in the tail. In order to define with greater precision, the molecular requirements for IgM polymer assembly, we have isolated several mutant hybridomas which produce monomeric IgM. For three such mutants, we synthesized cDNA clones of their mu mRNA and identified a mutation in the mu-chain which was responsible for the failure to assemble polymers. Mutant 205 has a 2-bp deletion which results in a termination codon after amino acid 556, effectively deleting the last 20 amino acids of the mu-chain. In conjunction with earlier reports, this result shows that the tail plays some role in assembly other than providing Cys-575, the penultimate amino acid, for disulfide bond formation. Both mutant 21 and mutant 201 have an A to G transition, which results in Tyr-455 in the fourth constant domain being replaced by a cysteine. We conclude that the integrity of both the C mu 4 domain and the 19 amino acid tail are required for the mu H chain to be assembled into polymeric IgM.

Amino Acid Sequence↗

PubMed1989

Structural requirements for IgM assembly and cytolytic activity. Effects of mutations in the oligosaccharide acceptor site at Asn402.

Glycosylation of IgG occurs at asparagine 297 of the gamma H chain and is necessary for the normal capacity of IgG to activate the classical pathway of complement-dependent cytolysis. IgM is glycosylated at five sites in the constant region of the mu H chain, of which glycosylation at asparagine 402 seems analogous to the glycosylation of IgG. In order to assess the importance of glycosylation at asparagine 402 for IgM cytolytic activity, we have used site-directed mutagenesis to produce IgM which is not glycosylated at this position. In particular we have tested the effects of substituting Gln for Asn 402 and Thr-Gly for Gly 403-Thr 404 in the third constant region domain. We tested the effects of these substitutions by expressing the mutant mu genes in hybridoma cells which produce the hapten-specific kappa-chain. The normal mu-chain is glycosylated at Asn 402, and, as expected, these mutations appear to abrogate glycosylation of the mutant mu-chains at position 402 and do not affect the hapten affinity of the IgM. However, both of these mutations cause the increased production of monomeric rather than polymeric IgM: the ratio of monomeric to polymeric IgM is 0.21, 3.5, and 10.3 for wild-type IgM, IgM-Gln 402, and IgM-Thr 403-Gly 404, respectively. The wild-type and mutant polymeric IgM preparations were compared for their capacity to promote complement-dependent cytolysis: IgM-Gln 402 and IgM-Thr 403-Gly 404 have approximately 31% and 4%, respectively, of the capacity of wild-type IgM.

Amino Acid Sequence↗

PubMed1989

Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.

The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis. Whereas normal polymeric IgM is composed predominantly of pentameric and hexameric molecules, the mutant IgM-Ser414 is covalently assembled as pentamers and smaller forms; IgM-Ser575 is assembled as covalent hexamers. IgM-Ser337 appears to include the same pentameric and hexameric forms as normal IgM except that, unlike normal polymeric IgM, most pentameric/hexameric IgM-Ser337 is not covalently assembled. J chain is present in polymeric IgM-Ser337 but absent in polymeric IgM-Ser414 and IgM-Ser575. IgM-Ser414 is defective in activating the classical pathway of complement dependent cytolysis. Our observations are consistent with models in which the covalent linkages between mu chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but indicate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Differential glycosylation of polymeric and monomeric IgM.

A small fraction of normal IgM is secreted as monomers rather than polymers. We show here that the mu chains of monomeric IgM are glycosylated differently from the mu chains of polymeric IgM and are comparable in their glycosylation to the mu chains from mutant hybridoma cell lines which produce predominantly monomeric IgM. The difference in glycosylation between monomer and polymer mu chains is due to differences in the terminal processing of their oligosaccharides. The glycosylation of the mutant mu chains is not itself responsible for the block in IgM polymer formation.

1-Deoxynojirimycin↗

PubMed1989

IgM--molecular requirements for its assembly and function.

The conventional model of IgM structure depicts a unique, array of mu, L and J chains, held together by well-defined disulfide bonds and other interactions. Some, but not all, recent data support this model. Here Ann Davis and Marc Shulman review recent, as well as older, studies of IgM and consider their implications for our understanding of IgM structure and function.

Animals↗

PubMed1989

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes.

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.

Animals↗

PubMed1988

Expression of an immunoglobulin kappa light-chain gene in lymphoid cells using a bovine papillomavirus-1 (BPV-1) vector.

Bovine papillomavirus-1 (BPV-1) replicates extrachromosomally in certain murine cell lines, suggesting that vectors based on the BPV-1 replicon might provide a means of obtaining more uniform gene expression among independent transformants. We have tested such a vector for the expression in hybridoma cells of the immunoglobulin kappa light-chain gene, but found that the level of expression varies greatly among transformants. Our results also indicate that in these transformants the vector has probably been incorporated into chromosomal DNA.

Animals↗

PubMed1988

C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain.

We have examined a defect in complement activation in a mutant trinitrophenyl-binding pentameric murine monoclonal IgM which has serine replacing the proline normally found at position 436 in the protein. The mutant protein showed equivalent hapten binding but a 100-fold decreased ability to initiate complement-dependent lysis of trinitrophenyl-coupled erythrocytes at physiological ionic strength (mu = 0.15). C4b deposition mediated by the mutant protein was impaired to a similar degree. C1 bound by the mutant protein showed C1s to C1-s conversion, suggesting normal activation. When measured at reduced ionic strength (mu = 0.06), the C1 and C1q binding affinity of the mutant protein was approximately one-half that of the wild type. However, the C1 binding affinity of the mutant protein showed a greater dependence upon ionic strength such that at physiological ionic strength we estimate a 50-fold lower C1 binding affinity for the mutant molecule. Kinetic studies suggested that this difference in affinity was largely attributable to differences in association rates. In addition, a fixed proportion of the mutant molecules showed no C1 binding. We conclude that the defect in complement activation occurs at the level of C1 binding. Our data support a role for the C mu 3 domain (residues 340-440) in C1 binding by IgM.

Complement Activating Enzymes↗

PubMed1988

Complement activation is required for IgM-mediated enhancement of the antibody response.

The ability of IgM antibodies to specifically enhance the thymus-dependent humoral immune response to particulate antigens is well documented. We have used two approaches to test whether complement factors play a role in this process. First, mice were depleted of C3 by treatment with cobra venom factor (CVF) and then immunized with SRBC with or without IgM-anti-SRBC. CVF treatment severely impaired the capacity of IgM to induce an enhanced anti-SRBC response. Moreover, it was shown that IgM can potentiate the response in C5-deficient AKR mice, thus demonstrating that the complement factors acting before C5 are the crucial ones. A second test compared the enhancing properties of two monoclonal IgM-anti-TNP antibodies where, because of a point mutation in the mu chain constant region, one of the antibodies is impaired in its capacity to activate complement. We show that the mutant antibody lacks the enhancing properties of the wild-type IgM. Activation of C3 by IgM antibodies as well as localization of antigen in the spleen seem to be necessary steps in the IgM-mediated enhancement of antibody responses. Our data offer an explanation to the immunosuppression described in CVF-treated animals as well as the low humoral immune responses in certain hereditary complement deficiencies. It is suggested that IgM indeed has an important physiological function in enhancing antibody responses to foreign substances.

Animals↗

PubMed1988

On the structure of polymeric IgM.

The cysteine at position 575 of the immunoglobulin mu heavy chain is thought to provide the only disulfide bonds joining the monomer subunits of mouse polymeric IgM. The importance of this cysteine in the assembly of polymeric IgM was investigated by using site-directed mutagenesis to produce mu chains with serine at position 575. Thirty percent of the secreted mutant IgM was covalently assembled polymer implying that cysteines other than Cys575 can form inter-subunit disulfide bonds. The polymeric IgM lacked J chain, mediated complement-dependent cytolysis and appeared to have a higher molecular weight than conventional IgM pentamers, as judged by sucrose gradient sedimentation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. Electron microscopy revealed that the mutant IgM molecule contained six subunits. Wild-type IgM, while synthesized predominantly as a pentameric molecule, was assembled in at least two other forms, which were distinguished by their electrophoretic mobility. The apparently higher molecular weight forms of wild-type IgM include hexameric molecules which, like the hexameric mutant IgM, contained much less J chain that the pentameric form and were 20-fold more efficient at activating complement-dependent cytolysis.

Animals↗

PubMed1988

Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin.

Animals↗

PubMed1988

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.

Animals↗

PubMed1987

Complement activation by IgM: evidence for the importance of the third constant domain of the mu heavy chain.

We have isolated and analyzed the DNA encoding the mu heavy chain constant region of a mutant IgM which is defective in initiating complement-dependent cytolysis. By assaying the expression of mu genes which were constructed in vivo from mutant and normal gene segments, we have mapped the mutation into a 555-base pair segment. In this segment there is one nucleotide change, such that the mutant mu gene encodes serine rather than the normal proline at amino acid position 436 in the third constant domain. We have used site-directed mutagenesis to revert this mutation to the normal sequence and shown that this substitution results in the production of IgM with the normal phenotype.

Amino Acid Sequence↗

PubMed1987

Biological properties of chimeric antibodies. Interaction with complement.

Genetic engineering allows the production of chimeric immunoglobulins where the same variable region is expressed in conjunction with constant regions of different species. We have compared the capacity of mouse and chimeric (mouse variable region/human constant region) immunoglobulin M (IgM) to trigger complement-dependent lysis and to bind complement component C1. Guinea pig and human C1 were bound more efficiently by mouse IgM than by chimeric IgM, whereas rat and rabbit C1 were bound more efficiently by chimeric IgM. Comparable results were obtained for complement as measured by the lysis of sensitized erythrocytes. These results indicated that differences between the human and mouse mu heavy chain constant regions define structures that are important in the C1-IgM interaction. Furthermore, species-specific differences in C1 also influence this interaction.

Animals↗

PubMed1986

Gene transfer of immunoglobulin light chain restores heavy chain secretion.

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.

Animals↗

PubMed1986

A region of the immunoglobulin-mu heavy chain necessary for forming pentameric IgM.

In order to define the molecular requirements for IgM pentamer formation, we have isolated several mutant hybridomas which produce predominantly monomeric IgM. For one such mutant, 102, we synthesized a cDNA clone of its mu-mRNA, and found an in-frame 39-bp deletion, which thus encodes a mu-chain lacking amino acids 550-562, a region spanning the fourth constant domain and the tail of the mu-chain. To prove that this deletion is sufficient to block pentamer formation, we used site-directed mutagenesis to construct a mu-gene lacking these 39 bp, and have shown that the expression of this altered mu-gene results in the production of monomeric IgM.

Amino Acid Sequence↗

PubMed1986

Analysis of hybridoma mutants defective in synthesis of immunoglobulin M.

Hybridoma mutants defective in the expression of IgM have been analyzed by molecular and somatic cell hybridization techniques. The frequency of kappa light-chain mutants in the hybridoma PC7 was much higher than for other cell lines. In contrast to the mutations which we observed previously, the kappa mutants examined here resulted from complete or partial deletion of the kappa gene. Mutants defective in mu chain synthesis were of more diverse types including deletions, gross rearrangements, and more subtle changes. One mutant containing a cis-acting mutation resulting in reduced expression of the mu chain had an associated partial duplication of the mu gene, while others making low or undetectable levels of mu had no gross alterations in genetic structure. The usefulness of this approach to the study of gene structure and expression is discussed.

Animals↗

PubMed1986

Activation of complement by immunoglobulin M is impaired by the substitution serine-406----asparagine in the immunoglobulin mu heavy chain.

We have isolated and analyzed the DNA encoding the mu heavy chain constant region of a mutant IgM that is defective in initiating complement-dependent cytolysis. By assaying the expression of mu-chain genes that were constructed in vitro from mutant and wild-type gene segments, we have mapped the mutation into a 555-base-pair segment that spans part of the third and fourth constant region domains. In this segment there is one nucleotide change, such that the mutant mu-chain gene encodes asparagine rather than the normal serine at amino acid position 406 in the third constant domain. We have used site-directed mutagenesis to introduce a comparable mutation into the normal mu-chain gene and confirmed that this substitution causes the production of IgM with the original mutant phenotype. Evidence is also provided that the serine-406----asparagine substitution might cause the mutant mu chain to be abnormally glycosylated.

Base Sequence↗