Combinations Of Heavy Chains Research Articles

Fragmentation of a Monoclonal Antibody by Peroxotungstate.

Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate. A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry. Exposure of a mAb to 1.07-1070ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH5-7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidativedisulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin. Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.

A possible basis for major histocompatibility complex-restricted T-cell recognition

Four distinct T-cell antigen-receptor gene loci have now been identified and partly characterized: alpha, beta, gamma and delta. All of these loci can rearrange in an immunoglobulin-like fashion and express polypeptides that contribute to either alpha:beta or gamma:delta T-cell receptor-CD3 complexes. Surprisingly, the T-cell receptor (TCR) delta coding regions are located entirely, or almost entirely, within the TCR alpha locus and share at least some of the V region gene segments, thus at least partly linking the two different types of receptor heterodimers. Analysis of potential T-cell receptor diversity, particularly that of the delta chain, indicates a striking concentration of somatic polymorphism in the V-J junctional region of the two heterodimers, four to six orders of magnitude higher than similar calculations for immunoglobulin light- and heavy-chain combinations. In contrast, the number of possible V region combinations in T-cell receptors is one hundredth to one thousandth that of immunoglobulins. TCR alpha: beta heterodimers are known to recognize many possible fragments of antigens embedded in the peptide-binding clefts of a relatively small number of major histocompatibility complex (MHC) molecules. Thus it is attractive to speculate that the V-J junctional portions of both types of T-cell receptor contact peptide antigens, whereas the remaining diversity regions contact the MHC. This contention is supported by molecular modelling studies and has interesting implications for the evolution of antigen-receptor genes.

Immunological types of lymphoproliferative disorders in a cohort: a 4-year study.

Immunological testing of malignant cells and serum from most cases of lymphoproliferative disorders (LPD) allows the cell type to be characterised as of B, T or "null" lymphocyte origin. Regional differences in the incidence of neoplasms of these types have been reported. Furthermore, most published series have drawn cases from referral institutions rather than the general population. In order to determine the true incidence of a cohort we surveyed an entire population, that of Tasmania, an island state of Australia with a population of 410,000, during a defined period, the years 1977-1980 inclusive, for the occurrence of LPD. A total of 248 cases was discovered, made up of 133 cases of non-Hodgkin's lymphoma (NHL), 30 of chronic lymphocytic leukaemia (CLL), 18 of acute lymphoblastic leukaemia (ALL), 54 of multiple myeloma (MM), eight of macroglobulinaemia (MGA) and five others. We identified B lymphocytes by the presence of surface membrane immunoglobulin (Smlg) and their ability to rosette with mouse red blood cells, and T lymphocytes by their ability to rosette with sheep red blood cells. Laboratory testing was performed in 201 (81%) of the cases and characterisation of the cell of origin as of B, T or "null" type was successful in 158 (64%). Of these 158, 136 (86%) were B, 4 (3%) T, and 18 (11%) "null". On B cell subtyping by heavy and light chain lg analysis the Tasmanian series, compared with other reports, had an apparent paucity of B-CLL, MM and MGA of lambda subtype (57 k to 12 lambda, k:lambda ratio 4.8:1) and an unusual incidence of B-CLL with the double lg heavy chain combination M+G. Surveys of this type may help to point to environmental or other factors important in the aetiology of LPD.

Surface immunoglobulins on hairy cells of 55 patients with hairy cell leukemia.

Surface immunoglobulins (SIg) were determined on peripheral blood samples from 55 patients with hairy cell leukemia (HCL) and on hairy cells from spleen preparations of 14 of these 55 patients. The patterns of SIg for HCL was compared to the patterns on peripheral blood leukemic cells from 39 patients with chronic lymphocytic leukemia (CLL) and 15 patients with poorly differentiated lymphocytic (PDL) lymphoma. Of the 55 HCL patients, 42 could be scored for individual heavy and light chains; 16 had only IgG, 14 had two or three heavy chains, 7 had only IgD, and 5 cases had no SIg and were E-rosette negative. This pattern was different from the B-cell pattern in CLL and PDL where there were few cases of IgG alone (5%) and many cases of IgM alone (50%). Surface marker profile did not correlate with survival in any of the sub-groups tested. HCL appears to be a B-cell lymphoproliferative disease in greater than 90% of cases; many combinations of heavy chains with only a single light chain can be demonstrated.