kD Isoform Research Articles

Detection of autoantibodies to the 65-kD isoform of glutamate decarboxylase by radioimmunoassay

Autoantibodies (AAb) to glutamate decarboxylase (GAD) occur with a high prevalence in sera of newly diagnosed type I (insulin-dependent) diabetic patients. The aim of this study was to establish a GAD-AAb radioimmunoassay using 125I-labelled GAD65 and to evaluate this assay in a cross-sectional study with newly diagnosed type I diabetic patients (diabetes duration < 6 weeks). Furthermore, subjects at high risk of developing type I diabetes and individuals suffering from other autoimmune diseases were examined in this assay. For GAD-AAb detection, 125I-labelled GAD65 was incubated with 10 microliters of human serum overnight on ice. Thirty of 51 (59%) type I diabetic patients but none of the 54 healthy blood donors tested were found to be positive. A displacement step using 100,000 g supernatant from rat brain containing or not containing GAD showed the specificity of the binding of 125I-GAD65. Concerning the individuals at high risk of developing diabetes. 9/12 (75%) islet cell antibody (ICA)-positive non-diabetic and 4/34 (12%) ICA-negative subjects with metabolic abnormalities were GAD-AAb positive. These results show the association between type I (insulin-dependent) diabetes mellitus and the occurrence of GAD65-AAb, which possibly predicts a risk of developing the disease.

EXPRESSION OF NK-ASSOCIATED SURFACE-ANTIGENS CD-56 AND CD-16 ON AML-CELLS

Monoclonal antibodies Leu 11a (CD16) and Leu 19 (CD56) were tested for reactivity with cells from 36 patients with acute myelogenous leukemia (AML) using two-colour flow cytometry. Blast cells were identified by a broad panel of monoclonal antibodies. In 33% (12/36) the monoclonal antibody Leu 19, which has been demonstrated to bind to the 140 kD isoform of the human neural cellular adhesion molecule N-CAM, found on peripheral natural killer (NK)-cells, neuroectodermal cells, activated T-cells, and myeloma cells, was shown to bind strongly to the leukemic cells. The monoclonal antibody Leu 11a, which recognizes a surface differentiation antigen associated with the low affinity FcRIII for IgG, expressed on NK-cells, granulocytes and macrophages were found to bind to leukemic cells of four of the 12 Leu-19 positive cases. 50% (6/12) of Leu-19 positive patients were classified as having M4 according to the French-American-British (FAB) morphology criteria. The potential diagnostic and clinical importance of CD 56 and/or CD 16 expression in acute myelogenous leukemia is presently under investigation.

Expression of the neural cell adhesion molecule and polysialic acid during early mouse embryogenesis.

The expression of the neural cell adhesion molecule (N-CAM) and alpha 2-8 linked polysialic acid (PSA), which is believed to be predominantly expressed on N-CAM, was investigated during early embryonic development of the mouse (embryonic days 7.5 to 10.0). By immunocytochemistry, in tissue sections, N-CAM and PSA were not detectable at embryonic day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonic day 8.0 N-CAM and PSA immunoreactivities were always predominantly associated with the plasma membrane. No tissue could be detected which was positive for PSA but negative for N-CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA-negative 180 and 140 kD isoforms of N-CAM were present at embryonic day 8.0 and strong expression of PSA-bearing, heavily sialylated N-CAM was not detectable before embryonic day 10.0. In Western blot analysis of N-CAM immunoaffinity purified from whole embryos and digested with neuraminidase as well as in Northern blot analysis, the 120 kD isoform of N-CAM or its corresponding mRNA were not expressed in detectable amounts during the time period investigated.

Myelin-associated glycoprotein is phosphorylated by protein kinase C.

The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of 32P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol ester phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2-3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [gamma-32P]ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes.

Distribution of growth hormone isoforms in sera from women with normal ovarian function, galactorrhea, and normoprolactinemia

To demonstrate if GH concentrations and molecular heterogeneity of GH correlates with the presence of galactorrhea in normoprolactinemic women with normal ovarian function. Aliquots of sera from women with normal ovarian function and normoprolactinemic galactorrhea were subjected to gel filtration chromatography, and the fractions were assayed for GH by the use of radioimmunoassay. Molecular weight of isoforms was calculated on a calibration curve obtained with molecular markers. The molecular variants were characterized on the basis of elution volume, molecular weight (MW), and partition coefficient. Basal serum GH levels were moderately elevated in all six normoprolactinemic women exhibiting galactorrhea. Chromatographic study of sera from these normoprolactinemic women showed the predominance of 40 to 50 kd molecular forms of GH as well as some very low MW GH isoforms. This pattern was different from that obtained in sera from normal women without galactorrhea who presented a predominance of heavier (> 60 kd) isoforms eluted before the GH labeled standard. The monomeric forms were present in less proportion but there was no significant difference as compared with galactorrheic group. Our investigation demonstrated elevated GH basal serum levels is normoprolactinemic women with galactorrhea, and chromatography in gel showed a low proportion of the large MW GH variants associated with a higher proportion of the dimeric forms and very low MW forms of GH. This is different from normal women without galactorrhea who had a predominance of heavier MW GH variants and lesser proportion of < 16 kd isoforms. It is concluded that an increased GH secretion may be responsible for abnormal lactation despite normal PRL levels in some women with normal ovarian function.

Alteration of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Activities by Site-Directed Mutagenesis

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.

Expression of the neural cell adhesion molecule (NCAM) on the haemopoietic cell line NALM-16

The Nalm-16 cell line was originally described as being of the null acute lymphoblastic leukaemia (ALL) phenotype. Using phenotypic and genotypic markers, we have demonstrated the line carries markers associated with cells of the B lineage. In addition, Nalm-16 binds a series of monoclonal antibodies characterized as predominantly recognising neuroectodermal tissues. Amongst these antibodies, UJ13A, 5.1.H11 and ERIC-1 have been shown to recognise the neural cell adhesion molecule (NCAM). Expression of the NCAM molecule is highly complex, with several isoforms of the protein resulting from the differential splicing of the NCAM mRNA transcript. Western-blot analysis of Nalm-16 cell extracts indicates that cells express the heavily polysialylated form of the molecule found on many embryonic tumours. Neuraminidase digestion indicates that the 140 kD isoform is predominantly expressed on Nalm-16, although the 120 kD isoform is present to a lesser degree. These findings have been confirmed using Northern-blot analysis.

Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons.

Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes. The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line. Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.

Initial characterization of the carbohydrate structure of MCP-1

MCP-1 is the principle monocyte chemoattractant produced by tumor cells in vitro. We previously identified different isoforms of MCP-1. Here we report that two predominant isoforms of MCP-1 (13 kD and 9 kD) can be distinguished by lectin binding. The results indicate that the disaccharide galactose-β1-3D-N-acetyl galactosamine is present on the 13 kD MCP-1 isoform but not the 9 kD isoform. Glycosylation of the 13 kD MCP-1 isoform does not appear to significantly change its antigenic properties and also does not affect its capacity to induce monocyte migration.

Characterization of myelin basic protein charge isomers from adult mouse brain

ADULT mouse MBP charge isomers (C1 or component 1, C2 or component 2, etc.) were purified from an acid soluble brain protein fraction by cation exchange chromatography. They were characterized by their elution profiles, their migration rates in alkaline-urea gels and by SDS-PAGE. Mouse C1 and C2 were both 14 kD in size, while C3, C4 and C5 consisted of the 18.5, 17 and 14 kD isoforms. Comparison of mouse and human MBP charge isomers showed that they were similar in that they both showed extensive charge heterogeneity, but different in that mouse MBP charge isomers were more cationic than their human counterparts. Finally, a possible explanation for the presence of the 14 kD MBP isoform in mouse myelin was suggested.

Discrete subpopulations, defined by CD45 isoforms, coexist within the leukaemic cells of B-chronic lymphocytic leukaemia patients

The expression of CD45 isoforms by B-CLL leukaemic lymphocytes has been analysed by 2 colour flow cytometry and vectorial iodination. The cytometry has demonstrated the presence of cells with different CD45 phenotypes which vary in the relative expression of the CD45RA and CD45RO determinants. Discrete populations have been detected which can coexist within an individual patient. One of these populations which is CD45RO-negative consists of cells expressing only the 230 kD isoform, in the others the smaller isoforms are expressed, the appearance of the CD45RO determinant of 180 kD being accompanied by the appearance of the 190 kD isoform. The relative proportion of cell populations is stable within patients but can be altered by phorbol ester which enhances CD45RO expression and diminishes CD45RA expression. The populations may be partially resolved by the density gradient fractionation.

High-molecular-weight Ly-5 isoforms expressed on T cells: activation-dependent expression

Interleukin 2 (IL-2)-dependent granular T lymphocyte (GTL) lines derived from murine spleen were shown to express a Ly-5 antigen with the so-called 'B220-epitope' recognized by 6B2 mAbs, which were normally exhibited on B-lineage cells. Immunoprecipitation analysis revealed that the Ly-5 isoform on GTL lines represented the highest-molecular-weight Ly-5 so far described (260 kd), distinct from the B220 isoform on a pre-B cell line (240 kd). The size difference between them was also evident after the endoglycosidase treatment (210 versus 190 kd), strongly suggesting that these Ly-5 isoforms had core proteins of distinct size. Sequential immunoprecipitation by 6B2 and 14.8 mAbs further indicated that the 260 kd Ly-5 on GTL lines predominantly expressed '6B2 epitope' while '14.8 epitope' dominated in the B220 (240 kd) on a pre-B cell line. Northern blot analysis of the Ly-5 transcripts using probes for the known alternative exons failed to show any evidence for mRNA of the 260 kd Ly-5 distinct from that of B220. Polymerase chain reaction analysis, however, suggested that the 260 kd isoform mRNA might be transcribed from a distinct promoter with a yet undefined exon(s). The 6B2+ Ly-5 isoform was hardly detected on normal splenic T cells, but was shown to be induced rapidly on the majority of T cells following IL-2 stimulation in vitro, indicating that this particular Ly-5 isoform behaved as an 'activation antigen' on T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the oligodendrocyte marker 2′3′‐cyclic nucleotide 3′‐phosphodiesterase in non‐glial cells

The 46 kD isoform of the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPI) was expressed in HeLa cells by transfection of its cDNA clone. The distribution of this polypeptide as mapped by indirect immunofluorescence and conventional epifluorescence microscopy appeared diffuse and generally uniform throughout the cytoplasm. Confocal microscopic imaging and analysis of pseudocolored images confirmed this distribution but also revealed that there was a high concentration of CNPI near the plasma membrane of the cell. This pattern is very similar to that observed by immunoelectronmicroscopy of myelinating oligodendrocytes (Trapp et al.: J Neurochem 51:859-868, 1988; Braun et al.: J Neurosci 8: 3057-3066, 1988). These results suggest that CNP may interact with a membrane-associated molecule that is not unique to oligodendrocytes.