125I Surface Labeling Research Articles

Evidence for the expression of HLAA-C class I mRNA and protein by human first trimester trophoblast.

In this study, evidence is provided that normal human first trimester extravillous trophoblast expresses class I HLA-C molecules in addition to HLA-G. cDNA from highly purified trophoblast cells obtained by flow cytometric sorting was amplified by reverse-transcriptase PCR using HLA locus-specific primers. The identity of the product was confirmed by Southern blotting and hybridization by a second HLA-C-specific oligonucleotide. HLA-C mRNA was clearly demonstrated in all trophoblast samples as well as in JEG-3 and BeWo choriocarcinoma cells. JAR choriocarcinoma cells did not express HLA-C. The presence of HLA-C protein in extravillous trophoblast was investigated using a panel of Abs: L31 is specific for heavy chains of all HLA-C alleles; Q1/28 reacts with all HLA class I products except HLA-G; HC-10 has preferential reactivity with HLA-B and HLA-C heavy chains. We performed 35S metabolic and 125I surface labeling of normal first trimester trophoblast and found abundant HLA-C intracellularly together with low levels of expression of both the beta 2m-associated forms and free heavy chains on the surface. Flow cytometric analysis of normal trophoblast confirmed the expression of a class I HLA molecule distinct from HLA-G by positive reactivity with Q1/28. Immunohistologic studies of first trimester placenta and the implantation site clearly showed expression of HLA-C in all extravillous trophoblast populations. Our results demonstrate the presence of two HLA class I molecules, HLA-G and HLA-C, on the surface of extravillous trophoblast. These results have implications in understanding how maternal uterine lymphocytes, notably the abundant NK-like cells, might recognize the implanting placenta.

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa)

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.

Differentiation-inducing cytokine P48 exists in a membrane-associated form.

P48 is a recently described 48-kDa differentiation-inducing cytokine isolated from the culture medium of the human leukemia line Reh. P48 induces differentiation and cytolytic activity in the promyelocytic cell line HL-60, and stimulates the release of TNF-alpha and IL-1 from peripheral blood monocytes. In further studies designed to examine the biosynthesis and function of P48, surface immunofluorescence flow cytometry analysis as well as 125I surface labeling and immunoprecipitation, revealed the presence of P48 on the surface of Reh cells. Triton X-114-treated Reh cells were partitioned into detergent and aqueous phases and separated by SDS-PAGE. Western blot analysis revealed that P48 partitioned exclusively into the detergent phase, suggesting an integral membrane association. Reh cells fixed with paraformaldehyde, but not K562 or P815, were able to stimulate the release of TNF-alpha from peripheral blood monocytes in a manner similar to that of secreted P48. Isolated plasma membranes from Reh cells could also stimulate TNF-alpha release. This TNF-alpha-releasing activity could be removed from detergent solubilized Reh membranes by immunoaffinity chromatography on an anti-P48 column. This study suggests that, in addition to being secreted into the culture medium, P48 is expressed on the surface of Reh cells in a biologically active form. The membrane form of P48 may be 1) a final maturation step before secretion or 2) a cell membrane-associated form that may be analogous to the membrane forms of TNF-alpha and IL-1.

Normal ADP-Induced Aggregation and Absence of Dissociation of the Membrane GP IIb-IIIa Complex of Intact Rat Platelets Pretreated with EDTA

ADP-induced platelet aggregation requires the presence of external calcium and fibrinogen. When human platelets are incubated for 30 min at 37 degrees C with 5 mM EDTA and then resuspended in a calcium containing medium, they lose their ability to bind fibrinogen and to aggregate in response to ADP stimulation. Under these conditions, the effect of EDTA is irreversible and accompanied by dissociation of the glycoprotein (GP) IIb-IIIa complex into its free subunits, GP IIb and GP IIIa. We studied the effect of incubation of intact rat platelets with 5 mM EDTA at 37 degrees C from 30 to 120 min. EDTA treated rat platelets showed normal aggregation in response to 5 microM ADP in the presence of added purified rat fibrinogen and bound 125I-labeled rat fibrinogen at the same rate and magnitude after stimulation with 5 microM ADP as untreated platelets. Control and EDTA treated rat platelets, labeled or not with 125I and solubilized in Triton X-100, had a similar pattern of immunoprecipitates after crossed immunoelectrophoresis (CIE) analysis. The rat GP IIb-IIIa arc was located by incorporation of an 125I-labeled polyclonal anti-human GP IIb-IIIa antibody. In contrast, in experiments using rat platelet lysates, we demonstrated that the rat GP IIb-IIIa is a Ca(2+)-dependent heterodimer as it was dissociated by EDTA. Using SDS-PAGE and two-dimensional SDS-PAGE, the rat GP IIb-IIIa complex was found to have characteristics similar to the human complex with the exception that the light chain of the rat GP IIb was undetectable after 125I surface labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C.

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.

Identification of the Major Glycosyl–Phosphatidylinositol Anchored Proteins on the Surface of Human Neutrophils

A novel mechanism of protein attachment to cell membranes involving the covalent linkage of the protein through an oligosaccharide to phosphatidylinositol has recently been defined. Many proteins that are anchored to the cell membrane by this mechanism can be released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Monoclonal antibodies are useful as probes in the study of the roles of cell-surface components in neutrophil function. Many monoclonal antibodies that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III (CD15 antibodies). Human neutrophil surface proteins identified by 125I surface-labeling using lactoperoxidase were examined for PI-PLC sensitivity, to identify the major surface proteins of human neutrophils that are anchored by a glycosyl-phosphatidylinositol linkage. The major surface-labeled protein identified by lactoperoxidase-catalyzed iodination was a approximately 68-90-kDa protein. Three major surface proteins identified by 125I-surface labeling of approximately 68-90, 57, and 33-kDa were released by PI-PLC treatment. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with 125I revealed a previously unidentified 98-115-kDa protein specifically reactive with CD15 antibodies that was released from the cell by treatment with PI-PLC. The roles of these proteins in neutrophil function remain to be determined.

Identification of Sporozoite Surface Proteins and Antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and 125I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.

Identification of outer oocyst wall proteins of three Cryptosporidium (Apicomplexa: Cryptosporidiidae) species by 125I surface labeling

Autoradiography of oocyst wall surface proteins of three Cryptosporidium spp. revealed common bands at 285 to 290, 145 to 148, 120, 57, and 32 kilodaltons (kDa). Cryptosporidium baileyi and C. muris share proteins at 180, 100, 80 to 81, 29, and 18 to 19 kDa; C. baileyi and C. parvum share one protein at 46 to 47 kDa; and C. muris and C. parvum share a protein at 67 to 69 kDa. Additional protein bands, each unique to one species, were also observed.

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji.

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.

Protein antigens of genital mycoplasmas.

Surface structures of the genital mycoplasmas Ureaplasma urealyticum and Mycoplasma hominis that are important in the human immune response and pathogenesis of disease are relatively poorly defined. In this study, an unusual antigen complex of U. urealyticum consisting of multiple bands forming a "ladder" pattern after electrophoretic separation was noted. It is similar to the variable V-1 surface antigen of Mycoplasma pulmonis. Data on U. urealyticum are only preliminary, but the ureaplasma antigen, if it proves to be analogous to V-1, may provide the antigenic determinants for distinguishing among serovars or serogroups and correlating them with pathogenicity. Surface proteins of M. hominis were identified with use of 125I surface labeling, [35S]methionine metabolic labeling, and immunoadsorption of rabbit antiserum. Comparison of M. hominis reference strains PG-21 and 4195 showed little homology between surface proteins, although with metabolic labeling they appeared essentially identical. Immunoblotting with patients' sera, using PG-21 as antigen, showed that most reactions were directed to surface proteins and that a 102K antigen (MH1) was recognized by 94% of the sera. MH1 was one of the few surface proteins of PG-21 that appeared to have counterparts in the other six reference strains, making MH1 a prime candidate for reliable and specific detection of M. hominis infection.

Biosynthesis and processing of the human insulin receptor.

We investigated the biosynthesis of the human insulin receptor in IM-9 lymphocytes and HEP-G2 hepatoma cells. Cells were first pulse labeled for 15 min with [35S]methionine and then chased for up to 4 h. At each time, the cells were solubilized in 1% Triton X-100; the insulin receptor was immunoprecipitated and then analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6%) and fluorography. At 15 min, a major precursor protein of 190,000 Mr was precipitated. During the chase period, two smaller proteins became apparent, which evolved into two major species of 130,000 and 95,000 Mr, the mature alpha- and beta-subunits, respectively. When IM-9 cells were trypsinized after pulse chase, the alpha- and beta-subunits were completely digested, whereas the 190,000-Mr precursor was unaffected. 125I-surface labeling of cells, followed by immunoprecipitation, revealed the presence of only the alpha- and beta-subunits, indicating that only these two species were on the cell surface. To study this biosynthetic pathway, several inhibitors were used (tunicamycin, monensin, and swainsonine). These inhibitors revealed the following. The receptor is first synthesized as a 170,000-Mr protein that is cotranslationally N-glycosylated to yield a high-mannose 190,000-Mr precursor. This precursor is rapidly transported from the endoplasmic reticulum to the Golgi apparatus where it is cleaved into two subunits of 120,000 Mr (alpha) and 90,000 Mr (beta). These subunits then increase in molecular weight by processing of the high-mannose oligosaccharides to the low-mannose complex type. The two subunits then migrate to the cell surface where they function to transmit the insulin signal.

Identification of Glycosylated Protein Antigens of Treponema pallidum and Treponema phagedenis

Comparison of autoradiographies of intrinsically [ 35S] methionine and [ 14C] glucosamine labeled Treponema pallidum (Nichols strain) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed four glycosylated proteins with molecular weights 30,500, 33,000, 35,000, and 59,000. T. phagedenis (biotype Reiter) was comparatively investigated and showed only two glycosylated proteins with molecular weights 33,000 and 34,000. The at the first time in treponemes identified glycosylated proteins could be precipitated with homologous human antibodies and characterized as antigens. By comparison with 125I surface labeling of T. pallidum and T. phagedenis it is suggested that the glycosylated protein antigens are localized on the surface of these treponemes.

Differences in expression and exposure of promastigote and amastigote membrane molecules in Leishmania tropica.

The accessibility of particular Leishmania tropica promastigote (extracellular) and amastigote (intracellular) membrane molecules might be related to the relative abilities of the two stages to induce host immune responses. To examine the exposure of membrane antigens on resident macrophage-susceptible promastigotes and resident macrophage-resistant amastigotes, both stages were analyzed by polyacrylamide gel electrophoresis and immunoblotting after specific labeling and extraction procedures. Protein compositional studies, using metabolic labeling of promastigotes and amastigotes, demonstrated that both forms possessed numerous endogenously synthesized proteins. In addition, a marked difference was revealed in the external exposure of promastigote and amastigote membrane constituents when analyzed by 125I surface labeling or Western blot analysis. Whereas nine promastigote proteins were intensely to moderately iodinated, only one amastigote membrane component was similarly labeled (9.5K band). Western blot analyses with serum from a rabbit immunized with a mixture of both L. tropica stages indicated that the majority of promastigote molecules accessible to 125I may also react with immune serum. However, Western blots of extracted amastigotes identified several bands not seen on radiographs and thus not accessible to 125I. The external exposure of these amastigote molecules was confirmed in that immune serum adsorbed with viable, intact amastigotes was no longer reactive with amastigote extracts. Further, by Western blot analyses of sodium dodecyl sulfate- but not Nonidet P-40-extracted amastigotes, three amastigote-specific membrane antigens not previously observed with nonionic detergent extraction methods were identified. The autofluorographic pattern of amastigotes intrinsically labeled with N-[3H]acetylglucosamine, an amino sugar which is incorporated into membrane carbohydrates, was in excellent agreement with the pattern of antigens reactive with antibody in Western blots. Thus, with these cell surface labeling and extraction methods, promastigote and amastigote membranes were shown to be significantly different. Amastigotes possessed several membrane-associated molecules, but few appeared to be either accessible or reactive with 125I. Moreover, the majority of molecules not reactive with 125I, but reactive with antibodies, may be glycosylated. These observations are discussed relative to the ability of amastigotes both to survive within the degradative milieu of macrophage phagolysosomes and to evade host immune reactivity.

Protein composition of the hyaline layer of sea urchin embryos and reaggregating cells

The protein composition of the sea urchin embryo hyaline layer has been studied by 125I surface labeling. The electrophoretic patterns of iodinated proteins indicate that the composition of the hyaline layer is species and stage-specific. Dissociation of iodinated embryos removes some labeled proteins of apparent molecular weights of 290,000, 175,000, 145,000, 110,000, 70,000, and 50,000. The electrophoretic pattern of labeled proteins found in the intact embryo is reestablished on the surface of aggregates following reaggregation. The possible function of these proteins with respect to cell adhesion, cell-sorting behavior and morphogenesis is discussed.

Relation between actin-associated proteins and membrane immunoglobulin in B-cells

The myosin-affinily technique, along with conventional immunoaffinity chromatography of membrane immunoglobulin (mIg). was used to study the relation between proteins that bind to actin, and those that co-isolate with the mIg of normal chicken B-cells. In the case of biosynthetically-labeled cells, we found approximately 13 actin-associated polypeptides. Of this group, eight could be labeled with 125I surface labeling. When the actin-associated proteins were compared to proteins that co-isolate with mIg during immunoaffinity chromatography, we found that two of them (mol. wts 55,000 and 34,000) co-isolate with mIg. The 55.000 mol. wt polypeptide can be labeled with 125I surface labeling techniques, while the 34.000 mol. wt protein cannot, suggesting that only the 55,000 mol. wt protein is exposed on the outer surface of the plasma membrane. It is speculated that the function of these proteins may be involved in linking mIg to actin and perhaps to the cytoskeleton.