Biomedical subjects

W A Jefferies

Publications and source records attributed to W A Jefferies.

At least 19 recordsLinked to original sources

PubMed2001

Iron metabolism in mammalian cells.

Most living things require iron to exist. Iron has many functions within cells but is rarely found unbound because of its propensity to catalyze the formation of toxic free radicals. Thus the regulation of iron requirements by cells and the acquisition and uptake of iron into tissues in multicellular organisms is tightly regulated. In humans, understanding iron transport and utility has recently been advanced by a "great conjunction" of molecular genetics in simple organisms, identifying genes involved in genetic diseases of metal metabolism and by the application of traditional cell physiology approaches. We are now able to approach a rudimentary understanding of the "iron cycle" within mammals. In the future, this information will be applied toward modulating the outcome of therapies designed to overcome diseases involving metals.

Animals↗

PubMed2000

Evidence of selective processing of immunodominant epitopes in virally infected cells.

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

TAP expression provides a general method for improving the recognition of malignant cells in vivo.

A major class of tumors lack expression of the transporters associated with antigen processing (TAP). These proteins are essential for delivery of antigenic peptides into the lumen of the endoplasmic reticulum (ER) and subsequent assembly with nascent major histocompatibility complex (MHC) class I, which results in cell surface presentation of the trimeric complex to cytolytic T lymphocytes. Cytolytic T lymphocytes are major effector cells in immunosurveillance against tumors. Here we have tested the hypothesis that TAP downregulation in tumors allows immunosubversion of this effector mechanism, by establishing a model system to examine the role of TAP in vivo in restoring antigen presentation, immune recognition, and effects on malignancy of the TAP-deficient small-cell lung carcinoma, CMT.64. To test the potential of providing exogenous TAP in cancer therapies, we constructed a vaccinia virus (VV) containing the TAP1 gene and examined whether VV-TAP1 could reduce tumors in mice. The results demonstrate that TAP should be considered for inclusion in cancer therapies, as it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic complement of the tumor or the MHC haplotypes of the host.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Melanotransferrin is produced by senile plaque-associated reactive microglia in Alzheimer's disease.

Melanotransferrin (MTf), also known as p97, has been localized in capillary endothelial cells of human brain. In Alzheimer's-diseased (AD) brain tissues, reactive microglial cells located in senile plaques exhibit elevated levels of MTf. The localization of the p97 protein may reflect its site of synthesis or could reflect a paracrine site of action. We examined the expression of MTf mRNA by in situ hybridization histochemistry using AD and healthy brain tissues. We also examined normal liver tissues by immunohistochemistry and in situ hybridization. In all the brain tissues examined, capillaries had positive signals for MTf mRNA. In AD tissues, expression of MTf mRNA appeared in reactive microglial cells in the grey matter specifically associated with dense plaques. In liver tissues, immunohistochemistry using anti-p97 antibody demonstrated that sinusoids were positively stained. In addition, in situ hybridization histochemistry revealed that hepatocytes had positive signals. These results suggest that p97 expression in reactive microglial cells are closely related to AD pathology. These results also support the notion that p97, which appears elevated in the cerebral spinal fluid and serum of AD patients, originates in the reactive microglia associated with dense senile plaques. Thus, p97 is a unique cellular hallmark of AD and further suggests that metal transport mechanisms play a role in this disease.

Aged↗

PubMed1999

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.

Amino Acid Sequence↗

PubMed1998

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers.

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1998

Surrogate antigen processing mediated by TAP-dependent antigenic peptide secretion.

MHC class I proteins assemble with peptides in the ER. The peptides are predominantly generated from cytoplasmic proteins, probably by the action of the proteasome, a multicatalytic proteinase complex. Peptides are translocated into the ER by the transporters associated with antigen processing (TAP), and bind to the MHC class I molecules before transport to the cell surface. Here, we use a new functional assay to demonstrate that peptides derived from vesicular stomatitis virus nucleoprotein (VSV-N) antigen are actively secreted from cells. This secretion pathway is dependent on the expression of TAP transporters, but is independent of the MHC genotype of the donor cells. Furthermore, the expression and transport of MHC class I molecules is not required. This novel pathway is sensitive to the protein secretion inhibitors brefeldin A (BFA) and a temperature block at 21 degrees C, and is also inhibited by the metabolic poison, azide, and the protein synthesis inhibitor, emetine. These data support the existence of a novel form of peptide secretion that uses the TAP transporters, as opposed to the ER translocon, to gain access to the secretion pathway. Finally, we suggest that this release of peptides in the vicinity of uninfected cells, which we term surrogate antigen processing, could contribute to various immune and secretory phenomena.

ATP-Binding Cassette Transporters↗

PubMed1996

Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions.

Ly-49 is a family type II transmembrane proteins encoded by a gene cluster on murine chromosome 6. One member of this family, Ly-49A, is expressed by a natural killer (NK) cell subset, binds to class I major histocompatibility complex (MHC) molecules, and blocks the killing of target cells bearing the appropriate H-2 antigens. Here we show that another member of this family which is expressed by an NK cell subset, Ly-49C, recognizes H-2b and H-2d structures which are distinct from and overlapping with those recognized by Ly-49A. Interactions between Ly-49A and C and their class I ligands are entirely blocked by the antibodies 5E6, YE1/48, YE1/32, and A1, all of which were found to recognize epitopes contained within the carbohydrate recognition domain (CRD). However, cell-cell binding assays revealed that class I binding specificity is conferred by a combination of sequences within both the CRD and a 19-amino acid adjacent region. We also investigated the question of whether Ly-49A and C form dimers on cells which express both receptors. When coexpressed on COS cells, sequential immunoprecipitation demonstrated that these receptors pair exclusively as homodimers, with no evidence for heterodimeric structures. These observations provide insight into both the biochemical nature of the Ly-49 family as well as the receptor functions of Ly-49C on NK cells.

Amino Acid Sequence↗

PubMed1996

Reactive microglia specifically associated with amyloid plaques in Alzheimer's disease brain tissue express melanotransferrin.

Several investigations have implicated the involvement of metals in neuropathologies. In particular, the disruption of iron metabolism and iron transport molecules have been demonstrated in Alzheimer's disease (AD). We have identified a novel pathway of iron uptake into mammalian cells involving melanotransferrin, or p97, which is independent of the transferrin receptor. Here we investigated whether there is a possible link between this molecule and the pathology of AD. The distributions of melanotransferrin, transferrin and the transferrin receptor were studied immunohistochemically in brain tissues from AD cases. In brain tissues from AD, melanotransferrin and the transferrin receptor were highly localized to capillary endothelium, while transferrin itself was mainly localized to glial cells. In brain tissue derived from AD patients, melanotransferrin was additionally detected in a subset of reactive microglia associated with senile plaques. Our demonstration that melanotransferrin mediates iron uptake through a pathway independent of the transferrin receptor indicates that this mechanism may have a role in AD.

Aged↗

PubMed1996

Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium.

One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.

Animals↗

PubMed1996

The macrophage cell surface glycoprotein F4/80 is a highly glycosylated proteoglycan.

Molecules whose expression is limited to particular leukocyte populations are of interest since they may perform unique functions for these cells. We therefore examined the biochemical nature of the F4/80 molecule, which is expressed solely on macrophage and dendritic cell subpopulations. Our study clearly indicates that post-translational modifications, which can influence both a protein's structural and functional features, constitute a major component of the 160-kDa cell-surface F4/80 molecule. The F4/80 molecule is synthesized as a single polypeptide chain which acquires numerous intramolecular disulfide bonds and requires an extended time period (T1/2 = 60 min) for transport to an endoglycosidase H-resistant form. The F4/80 molecule contains extensive N-linked glycosylation which contributes approximately 40 kDa to the mature molecule. The N-linked carbohydrates are of the branched, complex type, containing repeating N-acetylglycosamine or N-acetyllactosamine units which mediate the reactivity of the F4/80 molecule with Datura stramonium lectin. O-linked glycosylation is also present and contributes approximately 10 kDa to the F4/80 molecule. Furthermore, the sialic acid modifications of the F4/80 molecule are primarily through alpha 2-6 linkages to galactose. Finally, we demonstrate that the F4/80 molecule is a proteoglycan modified by chondroitin sulfate glycosaminoglycans. In addition to clarifying the nature of the F4/80 molecule biochemically, these post-translational modifications have specific implications for molecular recognition processes. We conclude that the modifications of the F4/80 molecule may mediate cell-cell recognition, cell adhesion, or ligand binding independently of the F4/80 molecule protein core.

Amino Acid Sequence↗

PubMed1996

Pumping iron in the '90s.

The role o f iron in cell division, cell death and human disease has recently gained increased attention. The best studied process for iron uptake into mammalian cells involves traps ferrin and its receptor. This review discusses evidence supporting the existence of other routes by which iron can enter mammalian cells. Specifically, iron uptake by the cell-surface GPI-linked traps ferrin homologue, melanotransferrin or p97, is described and possible functions of this traps ferrin-independent pathway are proposed.

Journal Article↗

PubMed1996

Serum levels of the iron binding protein p97 are elevated in Alzheimer's disease.

Alzheimer's disease is a progressive and incurable disease whose prevalence increases dramatically with age. A biochemical marker for monitoring the onset and progression of the disease would be a valuable tool for disease management. In addition, such a marker might be used as an end point in clinical intervention protocols. Here we provide evidence that the soluble form of the iron binding protein p97 is found in elevated amounts in the serum of Alzheimer's patients compared with healthy controls. This biochemical marker has the potential for identifying subjects afflicted with the disease and possibly for monitoring the onset and longitudinal progression of the disease.

Adult↗

PubMed1995

A novel iron uptake mechanism mediated by GPI-anchored human p97.

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.

Animals↗

PubMed1995

Detection of restricted isoform expression and tyrosine phosphatase activity of CD45 in murine dendritic cells.

CD45 is a cell surface transmembrane tyrosine phosphatase. It is expressed as distinct protein isoforms via alternative splicing of exons 4, 5 and 6. In T and B lymphocytes, CD45 is thought to play a critical role in antigen-dependent signaling through their respective antigen receptor complexes. However, the isoform expression and enzymatic activity of CD45 in other leukocytes remains largely unknown. Here, we examine the isoform expression and phosphatase activity of CD45 in murine dendritic cells (DC). Flow cytometric double-labeling analysis and biochemical analysis of purified splenic DC CD45 demonstrate that DC express both the CD45RB and CD45R0 isoforms. Flow cytometric analyses of freshly isolated splenic DC and thymic DC also indicate the expression of CD45RB and CD45R0 on these DC populations. In addition, we find that purified splenic DC CD45 possesses a high level of intrinsic tyrosine phosphatase activity. These data therefore establish the restricted isoform expression pattern of CD45 in murine DC and demonstrate that cells lacking specific antigen receptor complexes have active tyrosine phosphatase activity associated with CD45.

Animals↗

PubMed1994

Comparison of cell lines deficient in antigen presentation reveals a functional role for TAP-1 alone in antigen processing.

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides bound to major histocompatibility complex class I antigens on the cell surface of virus-infected cells. It is believed that the majority of peptides originate from cytoplasmic degradation of proteins assumed to be mediated by the "20S" proteasome. Cytosolic peptides are then translocated, presumably by transporters associated with antigen processing (TAP-1 and -2), into the lumen of the endoplasmic reticulum (ER) where binding and formation of the ternary complex between heavy chain, beta2-microglobulin (beta 2m) and peptide occurs. In this study, we have analyzed and compared the phenotype of two mutant cell lines, the thymoma cell line RMA-S and a small lung carcinoma cell line CMT.64, in order to address the mechanism that underlies the antigen processing deficiency of CMT.64 cells. Unlike RMA-S cells, vesicular stomatitis virus (VSV)-infected CMT.64 cells are not recognized by specific CTL. Interferon gamma (IFN-gamma) treatment of CMT.64 cells restores the ability of these cells to process and present VSV in the context of Kb. We show that although CMT.64 cells express a low level of beta 2m, the recognition of VSV-specific CTL is not restored by increasing the amount of beta 2m synthesized in CMT.64 cells. In addition, we find that CMT.64 cells express moderate levels of Kb heavy chain molecules, but most of it is unstable and rapidly degraded in the absence of IFN-gamma treatment. We infer that the antigen processing deficiency does not lie at the level of beta 2m or Kb production. We find also that the mRNAs for both TAP-1 and -2 are present in RMA and RMA-S cells but are absent in uninduced CMT.64 cells. Upon IFN-gamma induction, both mRNAs are highly expressed in CMT-64 cells. In addition, we find that the low molecular mass polypeptides 2 and 7, and additional components of the proteasome are induced by IFN-gamma in CMT-64 cells. Finally, introduction of the rat TAP-1 gene in CMT.64 cells restores CTL recognition of VSV-infected cells. These results indicate that a TAP-1 homodimer may translocate peptides in the ER and explain partially the CMT.64 defect and the RMA-S phenotype. These findings link a dysfunction in the transport and/or generation of antigenic peptides to the capacity of tumor cells to evade immunosurveillance and provide a unique model system to dissect this phenomenon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol-anchored protein.

Melanotransferrin, also called p97, is a cell surface glycoprotein which was first described as a marker antigen for human melanoma cells. Although p97 has a striking structural similarity to human serum transferrin and lactoferrin, its function has not yet been determined. One feature that distinguishes p97 from the other members of the transferrin family is the presence of a stretch of 24 hydrophobic amino acids at the C terminus, previously assumed to form a proteinacious transmembrane domain. In this study, sensitivity to bacterial phosphatidylinositol-specific phospholipase C, biosynthetic labeling with [3H]ethanolamine, and partitioning in Triton X-114 are used to establish that p97 is expressed at the cell surface as a glycosylphosphatidylinositol-anchored protein. In addition, to gain insight into the intracellular transport of p97, biosynthetic transport studies were performed on a melanoma cell line. These studies resulted in the identification of an additional form of p97 which is found in the medium and which likely does not originate from an alternatively spliced form of the p97 mRNA. These findings, together with our recent observation of the co-localization of p97 and the transferrin receptor in brain capillary endothelium (W. A. Jefferies, M. R. Food, R. Gabathuler, S. Rothenberger, T. Yamada, and P. L. McGeer, manuscript submitted) raise important questions about the function of the two forms of p97 detected and the possible involvement of this protein in a cellular iron uptake mechanism that is independent from the transferrin/transferrin receptor system.

Alternative Splicing↗

PubMed1994

Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells.

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

Adhesins, Bacterial↗