Cell Surface Iodination Research Articles

Interview with Dr. Heikki Rauvala regarding Pivotal Advance: Analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin).

HR: In the mid 80s we were searching for neurite outgrowth promoting factors that would act on cortical neurons. This had been my major research goal for several years, since I had established my own lab after a postdoctoral experience in the US in 1979–1980. Not many such factors were known, and the most interesting were fibroblast growth factors (FGFs). They were shown to regulate neuronal development, and to bind strongly to heparin and heparan sulfates. We homogenized young rat brains, and fractionated extracts on a heparin column, where not many proteins would bind at high salt concentration. The assay was simple: we adsorbed protein fractions on tissue culture plates, seeded primary rat brain neurons on the plates, and watched for neurite extensions after 24 h. In fact, the project went very well: we obtained a good amount of a protein that migrated as a single band of 30 kDa in Coomassie gels, which was obviously different from FGFs and was very active. We called this protein p30, and we realized very early on that it binds tightly to cation exchange columns, and thus must contain many basic residues. This worried me a bit, since cationic proteins were known to be very common in the nucleus, where they bind DNA. So we raised antibodies against p30 and used them and lactoperoxidasecatalyzed cell surface iodination to label intact neuroblastoma cells and brain neurons. This satisfied me that p30 was expressed in the extracellular space, essentially as a membraneassociated protein. The purification of p30 was reported in The Journal of Biological Chemistry in 1987 [1]. The name amphoterin came later, when we realized that p30 had both positively and negatively charged segments.

Modulation of the major histocompatibility complex class II-associated peptide repertoire by human histocompatibility leukocyte antigen (HLA)-DO.

Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II-eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO(-) and DO(+) transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A(1), an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

Functional Expression of the Chicken Low Density Lipoprotein Receptor-related Protein in a Mutant Chinese Hamster Ovary Cell Line Restores Toxicity of Pseudomonas Exotoxin A and Degradation of α2-Macroglobulin

The low density lipoprotein receptor-related protein (LRP) is responsible for the clearance of several physiological ligands including a complex of proteinase and alpha2-macroglobulin (alpha2M) and for the entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP (designated LRP100) and two intermediates encoding 25 and 67% of the receptor (designated LRP25 and LRP67, respectively) using overlapping cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%, respectively, of LRP100 plus the transmembrane and intracellular domains. Transient transfection of these plasmids into COS-7 cells yielded recombinant proteins of expected molecular mass and immunoreactivity. However, LRP100 was incompletely processed into alpha- (515-kDa) and beta- (85-kDa) chains and was poorly transported from the endoplasmic reticulum to the Golgi compartment. Stable transformants of LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster ovary cell line that lacked expression of endogenous LRP and was resistant to PEA. All forms of recombinant LRP proteins were transported from the endoplasmic reticulum to the Golgi apparatus in Chinese hamster ovary cells as shown by their sensitivity to endoglycosidase H and resistance to neuraminidase. Cell surface iodination and subcellular fractionation studies indicated that all three LRP variants were expressed on the plasma membrane. Furthermore, expression of the three LRP variants restored, to various degrees, sensitivity to PEA and the ability to degrade methylamine-activated alpha2M (alpha2M*). These data suggest that deletion of large internal portions of LRP, including the processing site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with alpha2M* or PEA. This LRP expression system may allow for the characterization of domains within LRP responsible for its multifunctionality.

Effect of hypercarbia on surface proteins of cultured bovine endothelial cells.

Hypercarbia is a common complication of respiratory failure, and the technique of "permissive hypercapnia" is used to ventilate individuals with increased peak airway pressures on mechanical ventilators, resulting in elevated arterial PCO2. We studied the effects of hypercarbia on cultured bovine aortic and main pulmonary artery endothelial cell surface proteins, assessing cell surface iodination using lactoperoxidase bound to latex microspheres. We found that 4 h of exposure to 10% CO2 increased the display of substances of apparent molecular masses of 27, 47, and 52 kDa. This effect was not mimicked by acidotic media. Western blots of detergent extracts of main pulmonary artery endothelial cell monolayers did not show increased expression of carbonic anhydrase IV (molecular mass = 52 kDa) after incubation under hypercarbic conditions. Hypercarbia did not change the pattern of [35S]methionine incorporation into endothelial cell proteins. We conclude that hypercarbia of 4-h duration changes iodinated endothelial cell surface proteins. We speculate that this effect may be related to changes in secretion or display of apical cell membrane-associated proteins.

Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Identification of Fc alpha receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages.

One of the hallmarks of mucosal-host defense is the clearance of inhaled Ags by alveolar macrophages (AM) through interactions of IgA Abs and IgA Fc receptors (Fc alpha R). AM constitutively expressed Fc alpha R at lower levels than freshly isolated and in vitro-differentiated monocytes as determined by immunofluorescence using four anti-Fc alpha R mAb. SDS-PAGE analysis of iodinated cell surface proteins revealed that Fc alpha R on AM has an Mr of 50 to 65 kDa, slightly lower than that on monocytes (55-75 kDa). Treatment of AM Fc alpha R by N-glycanase gave rise to a protein core of 28 KDa, smaller than the 32-kDa backbone of blood monocytes. AM Fc alpha R molecules were unaffected by phosphatidylinositol-phospholipase C treatment. Fc alpha R transcripts were analyzed by reverse transcription-PCR using primers in the 5' and 3' regions of a U937 Fc alpha R cDNA. Three transcripts were amplified, cloned, and sequenced from AM and/or monocyte mRNA, the full length Fc alpha R and two alternatively spliced products corresponding to deletions of 66 and 288 nucleotides in the portion coding for the extracellular domain; they were named Fc alpha R a.1, a.2, and a.3, respectively. These PCR products were transcribed and translated in vitro into three proteins (Mr 32, 30, and 22 kDa, respectively), in which the 32- and 30-kDa species were immunoprecipitated by an anti-Fc alpha R mAb. The predicted size of the protein encoded by the Fc alpha R a.2 transcript without the leader peptide is Mr approximately 27,400, a value that is consistent with the Mr of AM Fc alpha R backbone. These results indicate that AM express at their surfaces a protein product of an alternatively spliced Fc alpha R transcript, the Fc alpha R a.2 isoform, that might have physiologic relevance in IgA-mediated host defense at mucosal sites.

Enhanced Release of Amyloid -Protein from Codon 670/671 Swedish Mutant -Amyloid Precursor Protein Occurs in Both Secretory and Endocytic Pathways

The mutation at codons 670/671 of beta-amyloid precursor protein (betaPP) dramatically elevates amyloid beta-protein (Abeta) production. Since increased Abeta may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimer's disease, evaluation of the cellular mechanism(s) responsible for the enhanced Abeta release may suggest potential therapies for Alzheimer's disease. In this study, we analyzed Chinese hamster ovary cells stably transfected with either wild type betaPP (betaPP-wt) or "Swedish" mutant betaPP (betaPP-sw) for potential differences in betaPP processing. We confirmed that increased amounts of Abeta and a beta-secretase-cleaved COOH-terminally truncated soluble betaPP (betaPPs) were secreted from betaPP-sw cells. As shown previously for betaPP-wt cells, Abeta was released more slowly than the secretion of betaPPs from surface-labeled betaPP-sw cells, indicating that endocytosis of cell surface betaPP is one source of Abeta production. In contrast, by [35S]methionine metabolic labeling, the rates of Abeta and betaPPs release were virtually identical for both cell lines. In addition, the identification of intracellular betaPPs and Abeta shortly after pulse labeling suggests that Abeta is produced in the secretory pathway. Interestingly, more Abeta was present in medium from betaPP-sw cells than betaPP-wt cells after either cell surface iodination or [35S]methionine labeling, indicating that betaPP-sw cells have enhanced Abeta release in both the endocytic and secretory pathways. Furthermore, a variety of drug treatments known to affect protein processing similarly reduced Abeta release from both betaPP-wt and betaPP-sw cells. Taken together, the data suggest that the processing pathway for betaPP is similar for both betaPP-wt and betaPP-sw cells and that increased Abeta production by betaPP-sw cells arises from enhanced cleavage of mutant betaPP by beta-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH2 terminus of Abeta.

Characterization of monoclonal antibodies to Yersinia enterocolitica iron-regulated proteins.

Yersinia enterocolitica 1165 (0:8) expressed several iron-regulated proteins with molecular masses of 240, 194, 80, 79, 70, and 67 kDa. These proteins were not detected in cells grown in iron-rich conditions. Cell surface iodination indicated that the 240- and 190-kDa proteins (HMWPs) were not surface exposed, whereas the 67- and 70-kDa proteins appeared to be exposed to the cell surface. Incubation with iron protected the 67- and 70-kDa proteins from proteinase K treatment, suggesting that they may be involved in iron acquisition. Monoclonal antibodies (MAbs) were produced against the HMWPs and the 67-kDa iron-regulated protein. MAbs to the HMWPs not only recognized the 240- and 194-kDa proteins but also reacted with the 67- and 70-kDa iron-regulated proteins. Similarly, MAbs to the 67-kDa protein reacted with the 67- and 70-kDa proteins and the HMWPs, suggesting that these iron-regulated proteins are related immunologically. In addition, the MAbs recognized the 67- and 70-kDa proteins and HMWPs from other Y. enterocolitica serotypes, suggesting that the antigenic sites recognized on these iron-regulated proteins are conserved. The MAbs examined did not inhibit iron binding or iron uptake and did not provide protection against a Y. enterocolitica 1165 (0:8) infection in a systemic mouse infection model. Although these MAbs were not protective in this model, these iron-regulated proteins may play a role in iron acquisition and virulence, but the MAbs examined are probably not directed against epitopes involved in iron acquisition or virulence.

Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts

The hyaluronan receptor CD44 is an abundant glycoprotein expressed on a variety of different cell types. In fibroblasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extraction. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton. In this study we examined the structural features of CD44 required for its Triton X-100 insolubility in murine fibroblasts. We expressed in L cells the wild-type hematopoietic form of CD44, a mutant CD44 lacking the cytoplasmic domain, and two mutant CD44 molecules with substituted transmembrane domains. Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined. No difference in detergent solubility was observed between wild-type and tailless mutant-transfected molecules. However, both CD44 mutants with a heterologous transmembrane domain, derived from either the CD3 zeta chain or CD45, were completely soluble in Triton X-100. These results demonstrate that the transmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells. No obvious colocalization of CD44 and actin stress fibers was observed before or after treatment with cytochalasin D, and no change in the detergent extraction profile of wild-type and mutant CD44 molecules was effected by cytochalasin D. In equilibrium density sucrose gradients the Triton-insoluble CD44 component was found in the low density fractions, indicating an association with Triton X-100-insoluble lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of insulin-like growth factor II on uptake of arylsulfatase A by cultured rat hepatocytes and Kupffer cells

Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocytes and Kupffer cells isolated from adult rat liver. Affinity labeling with [125]insulin-like growth factor II revealed a protein of Mr 250 000 in both cell types. Labeling was inhibited by an antiserum against the mannose 6-phosphate/insulin-like growth factor II receptor. In Kupffer cells, [125I]insulin-like growth factor II was also cross-linked to a second protein of Mr 130 000. In both cell types, insulin-like growth factor II was 10 times more potent than insulin-like growth factor I in displacing [125I]insulin-like growth factor II from its receptor. The mannose 6-phosphate-specific uptake of [125I]arylsulfatase A via the mannose 6-phosphate/insulin-like growth factor II receptor was inhibited by insulin-like growth factor II and antibodies against the receptor, but was not affected by insulin-like growth factor I, insulin or transforming growth factor β1. Cell surface iodination followed by immunoprecipitation of the mannose 6-phosphate/insulin-like growth factor II receptor showed that expression of the mannose 6-phosphate/insulin-like growth factor II receptors at the plasma membrane was increased two-fold by insulin-like growth factor II. These results suggest that binding of insulin-like growth factor II to the mannose 6-phosphate/insulin-like growth factor II receptor blocks the binding and uptake of mannose 6-phosphate-containing lysosomal enzymes and may be directly involved in a co-ordinate regulation of ligand uptake from plasma into hepatocytes and Kupffer cells.

Effects of Cytoplasmic Domain Length on Cell Surface Expression and Syncytium-Forming Capacity of the Simian Immunodeficiency Virus Envelope Glycoprotein

We previously reported that truncation of the terminal 146 amino acids of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced envelope-specific syncytium formation in HeLa T4, CEM × 174, and HUT 78 cell lines and caused a change in the conformation of the transmembrane subunit of the envelope complex on the surface of these cells [Ritter et al. (1993) Virology 197, 255-264; Spies et al. (1994) J. Virol. 68, 585-591]. To investigate the effects of different lengths of the cytoplasmic domain on syncytium formation and cell surface expression, we have compared the expression and cytopathic effects induced by five SIVmac239 envelope constructs which vary in the lengths of their cytoplasmic domains. In contrast to the envelope protein truncated by 146 amino acids, the ability of proteins truncated by 98 or 161 amino acids to form syncytia was substantially reduced in CEM × 174 and HUT 78 cells, white syncytium formation by a protein truncated by 53 amino acids was only slightly reduced compared to the full-length protein. Furthermore, only the glycoprotein which was truncated by 146 amino acids induced syncytium formation in HeLa T4 cells. When examining the expression of the truncated proteins on the surface of HeLa T4 cells, we found that, in contrast to the full-length SIVmac239 protein, each of the truncated transmembrane subunits could be efficiently biotinylated with the membrane impermeable reagent NHS-SS-biotin. Furthermore, using cell surface Iodination, we found stable oligomeric forms of both the transmembrane subunits and the uncleaved precursor proteins of each mutant protein on the surface of HeLa T4 cells. Using pulse-chase analysis, we also found that the precursor of the protein truncated by 98 residues was degraded more rapidly than the wild-type and the other mutant proteins. Finally, we constructed two mutants which expressed a full-length TM protein or a TM protein with a 146 amino acid C-terminal deletion and had most of the coding sequences of their SU subunits deleted. Neither of these two proteins was found to cause syncytium formation in HeLa T4, CEM × 174, or HUT 78 cell lines even though we could detect both proteins on the surfaces of HeLa T4 cells using iodination. These results could explain why the selection of truncated variants of SIV which emerge after prolonged passage in human cell lines is restricted to truncations which remove close to 146 amino acids of the cytoplasmic domain of the TM protein.

The effect of sphingosine on the release of fibronectin from human lung fibroblasts

Previous work from our laboratory (Scheidl et al. (1992) Biochim. Biophys. Acta 1135, 295–300) has shown that sphingosine (Sph), a known inhibitor of protein kinase C (PKC) inhibited the release of fibronectin (FN) into the media of fibroblast cells in culture and that this effect was independent of PKC. We now report that the action of Sph was time dependent (maximum inhibition in 2 h), concentration dependent (0.5–10 μM, at which concentration Sph was not toxic), and rapidly reversible after removal of Sph. Incubating with [ 35S]methionine, we found Sph reduced FN release either by inhibition of FN synthesis or FN secretion. To distinguish between these two possibilities, cells were treated with monensin, an inhibitor of FN secretion, which together with Sph showed no additive effect on FN release. Cycloheximide also inhibited FN release, but this inhibition was additive to that with monensin. We concluded that Sph inhibited secretion of FN, not synthesis. Furthermore, intracellular FN declined less in Sph-treated than in untreated control cells. By cell-surface iodination with Na 125I and lactoperoxidase, followed by immunoprecipitation with antibodies to FN and α 5 β 1-integrin, we showed that labeled multimeric and dimeric cell-surface FN declined, whereas integrins remained unchanged upon Sph treatment. This result suggested that though the number of cell-surface receptors for FN was not affected by Sph, their affinity may be reduced. In addition, we found that either pre- or co-treatment for 30 min with Sph caused a robust inhibition of cell adhesion to FN-coated plastic. We conclude that Sph rapidly inhibits FN secretion, lowers cell-surface FN, and greatly reduces cell adhesion to a FN substratum.

Immunochemical characterization and differentiation of two approximately 300-kD erythrocyte membrane-associated proteins of Plasmodium falciparum, PfEMP1 and PfEMP3.

Erythrocyte membrane-associated antigens of Plasmodium falciparum have been of long-standing interest as potential adherence receptors and vaccine candidates. We recently identified in trophozoite-stage infected erythrocytes a novel high molecular weight erythrocyte membrane-associated protein of P. falciparum, PfEMP3, defined by Western blotting with the rat monoclonal antibody 12C11. Genomic clone lambda 12.1.3 and cDNA clone p12.2 contain nucleic acid sequences encoding PfEMP3. Analysis of Malayan Camp strain parasites by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels revealed that PfEMP3, defined by Western blot, has the same relative molecular weight (M(r)) as the surface-exposed protein PfEMP1 defined by cell surface iodination. We show here that PfEMP3 is distinct from PfEMP1 by three criteria. First, 125I-labeled PfEMP1 was resolved from PfEMP3 by extended migration on 4% gels. Second, in two strains of P. falciparum in which 125I-PfEMP1 has a different M(r), PfEMP3 had the same M(r). Third, immunization studies were performed with fusion proteins derived from clones lambda 12.1.3 and p12.2. Although one rabbit, Rb 05.75, immunized with the PfEMP3-derived fusion protein beta gal12.1.3, produced a serum that strongly immunoprecipitated PfEMP1 as well as PfEMP3, most sera immunoprecipitated only PfEMP3. Furthermore, immunoprecipitation of PfEMP3 by Rb 05.75 serum was blocked by the glutathione S-transferase 12.1.3 fusion protein, whereas immunoprecipitation of PfEMP1 was unaffected. Therefore, we conclude that PfEMP1 and PfEMP3 are antigenically distinct.

Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 (Ly-1) and has been shown to induce B cell proliferation upon mAb binding. The serologically defined Ly-19.2 and Ly-32.2 lymphocyte alloantigens have mouse strain distribution patterns similar to that of the Lyb-2/CD72 alleles and map to the same region on chromosome 4 as Lyb-2/CD72. Our recent isolation of the Lyb-2a, -2b, and -2c cDNA has enabled us in this report to examine the relationship between Ly-19, Ly-32, and Lyb-2/CD72. A rat T cell line transfected with a mouse Lyb-2a cDNA is recognized by Ly-19.2-specific mAb, whereas transfectants expressing the Lyb-2b cDNA are recognized by both Ly-19.2 and Ly-32.2-specific mAb. Cell surface iodination immunoprecipitation analysis from Lyb-2a cDNA transfectants using Lyb-2a- and Ly-19.2-specific mAb as well as from Lyb-2b cDNA transfectants using Lyb-2b-, Ly-19.2-, and Ly-32.2-specific Ab, produced immunoprecipitates containing comigrating 45-kDa polypeptides. Preclearing studies with these transfectants indicate that the immunoprecipitated proteins represent the same polypeptide chain. These results demonstrate that the mouse Ly-19.2 and Ly-32.2 alloantigens are in fact the B cell differentiation Ag Lyb-2/CD72.

Identification of a novel subunit of the type I interferon receptor localized to human chromosome 21

Expression in mouse cells of the cloned human IFN alpha receptor (IFN alpha R) subunit selectively confers response and binding to human IFN alpha 8, indicating that other subunits are involved in IFN alpha binding. We report here that a new monoclonal antibody (mAb), termed IFNaR beta 1, recognizes a novel IFN alpha R subunit different from the one recently cloned and distinct from the alpha subunit recognized by the IFN alpha R3 mAb. The IFNaR beta 1 mAb blocks the biological effect of seven different Type I IFNs. Immunoprecipitations after cell surface iodination demonstrate that the IFNaR beta 1 mAb recognizes a protein with a molecular mass of 100 kDa in Daudi and U-266 cells that express normal IFN alpha R. However, a 55-kDa protein instead of the 100-kDa product was immunoprecipitated in the IFN alpha-resistant U-937 cell line that express the variant form of the receptor. We also demonstrate that the gene that codes for this novel IFN alpha R subunit maps to human chromosome 21, as do the cloned IFN alpha R subunit and the alpha subunit, indicating the existence of a locus on this chromosome that regulates binding for Type I IFNs.

A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.

A hamster mAb (10.1.1), raised against long term cultures of uncloned thymic stromal cells, selectively labeled a subpopulation of medullary stromal cells in situ. By ultrastructural and phenotypic criteria, the stromal cells labeled by this mAb were judged to be epithelial. Although some of the 10.1.1+ epithelial cells were reticular, others were globular and some were associated with structures resembling Hassal's bodies. Ultrastructural immunohistochemistry suggested that 10.1.1 labeling of some of the epithelial cells was preferentially associated at areas of epithelial cell contact with adjacent thymocytes. Reactivity of thymic stromal cells with this antibody was developmentally regulated. A few scattered 10.1.1+ cells were observed at day 14 of gestation, and there were progressive increases in both the extent and intensity of 10.1.1 labeling evident through birth. One thymic stromal cell line, Z210.1, exhibited low levels of constitutive reactivity with this antibody. Exposure to IL-1 resulted in enhanced 10.1.1 reactivity of this cell line, with little, if any, additional response to TNF-alpha or IFN-gamma. Under the same conditions, ICAM-1 expression by this cell line was elevated in response to IL-1, TNF-alpha, or INF-gamma. Immunoprecipitation of detergent lysates prepared from Z210.1.7 cells exposed to IL-1 24 h before cell surface iodination identified a cell surface protein with a molecular mass of about 92 kDa under nonreducing conditions and about 95 kDa under reducing conditions. Digestion of 10.1.1 immunoprecipitates with N-glyconase resulted in a small (5 kDa) reduction in molecular mass. The molecule recognized by the 10.1.1 mAb was distinct from ICAM-1, which possessed a molecular mass of 100 kDa (nonreduced) and 110 kDa (reduced), and also displayed a smaller N-glyconase-resistant molecular mass (65 to 85 kDa).

Cysteine-dependent zinc binding by membrane proteins of Giardia lamblia.

The abundant, highly variable surface proteins (VSPs) which cover the surface of Giardia lamblia trophozoites compose a group of extremely cysteine (C)-rich proteins in which more than half of the cysteines are in the motif CXXC. Because of the constancy of these features among the known VSPs and the prominence of cysteine and particularly CXXC in proteins that bind zinc and other metals, we asked whether G. lamblia VSPs bind zinc in vitro. VSPs are the major protein component of Triton X-114 detergent-phase extracts of G. lamblia trophozoites and can be readily identified by surface iodination of intact cells. The partitioning of 65Zn binding into the Triton X-114 detergent phase and the correspondence between surface iodination and zinc binding patterns of four G. lamblia strains or sublines with different VSPs support the idea that VSPs bind zinc. The requirement for renaturation of blots with a reducing agent indicates that Zn2+ is coordinated by cysteine residues, rather than by other amino acids. Binding did not appear to be specific to zinc since it was inhibited by competition with other divalent metal ions. The abundance of the VSPs and the prevalence of metal binding motifs among all known variants suggest that they may play an important role in trophozoite survival and colonization in the host.

Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins

Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well-accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase-catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water-soluble sulfo-NHS-biotin. Partitioning of the keratins to a soluble and an insoluble pool after "cell surface" 125I-labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti-keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques.

Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes.

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.