Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK-Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.     

CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.     

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.     

Chromosome sublocalization of a cDNA for human DNA polymerase-beta to 8p11----p12

We have localized a cDNA fragment that codes for human DNA polymerase-beta. Using somatic cell and in situ hybridization techniques, this cDNA was cloned by screening a human KM-3 cell cDNA library in lambda gt 11 for expression of fused beta-galactosidase-human DNA polymerase-beta proteins. We have mapped this human polymerase-beta gene to the short arm of chromosome 8 in the subregion 8p11----p12.     

Enhancement of human melanoma antigen expression by IFN-beta

Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines. We have now identified IFN-beta as an additional stimulus to TAA expression, including Melan-A/MART-1, gp100, and MAGE-A1. IFN-beta (but neither IFN-alpha nor IFN-gamma) augmented both protein and mRNA expression of melanocytic TAA in 15 melanoma lines (irrespective of initial Ag-expression levels). Treatment of low Ag melanoma lines with IFN-beta increased expression of melanocyte-lineage Ags, inducing susceptibility to lysis by specific CTLs. Treatment with IFN-beta also enhances expression of class I HLA molecules, thereby inducing both nominal TAA and the presenting HLA molecule. Data from fluorescent cellular reporter systems demonstrated that IFN-beta triggers promoter activation, resulting in augmentation of Ag expression. In addition to enhancing TAA expression in melanomas, IFN-beta also stimulated expression of the melanocytic Ag gp100 in cells of other neural crest-derived tumor lines (gliomas) and certain unrelated tumors. Because IFN-beta is already approved for human clinical use in other contexts, it may prove useful as a cotreatment for augmenting tumor Ag expression during immunotherapy.     

Structure of the AAA ATPase p97

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.     

A molecular model for the tumour-associated antigen, p97, suggests a Zn-binding function.

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C-lobe iron-binding cleft and the interlobe contact region. The latter includes within the N-terminal lobe a Zn-binding consensus sequence found in metallopeptidases, and in the C-terminal lobe a glutamic acid residue (Glu-394) capable of completing a potential thermolysin-like Zn-binding site. Thus, p97 may have a Zn-binding potential, unique amongst the transferrin superfamily.     

Mutational analysis of the human p53 gene in malignant melanoma

Nine metastatic melanoma cell lines and two melanocyte cell lines were analyzed for point mutations in highly conserved regions of the p53 gene. No mutations were detected in the two melanocytic cell lines and in eight melanoma cell lines. However, a C----T transition at codon 248, resulting in a substitution of tryptophan for arginine, was found in one melanoma cell line. On immunohistochemical staining, only this cell line showed reactivity for mouse monoclonal antibody 1801, which is immunoreactive with human p53 protein. The original paraffin-embedded specimen from which this mutant cell line was established was obtained, and sequence analysis detected the identical mutation in the p53 gene as that seen in the derived cell line. This is the first report indicating point mutations in the p53 gene in malignant melanocytic tissues.     

Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells

The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI-anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI-anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI-anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI-anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster ( approximately 88 kDa) than the native p97 ( approximately 95-97 kDa). The insect GPI-anchored p97 was sensitive to PI-PLC, which exposed a detectable cross-reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK-MEL-28) cells, whereas the Sf9 cell specific secretion rate was 10-fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI-anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI-anchored proteins.     

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and⁶⁷Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%–40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.     

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.     

Imbalances in p97 co‐factor interactions in human proteinopathy

The ubiquitin‐selective chaperone p97 is involved in major proteolytic pathways of eukaryotic cells and has been implicated in several human proteinopathies. Moreover, mutations in p97 cause the disorder inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD). The molecular basis underlying impaired degradation and pathological aggregation of ubiquitinated proteins in IBMPFD is unknown. Here, we identify perturbed co‐factor binding as a common defect of IBMPFD‐causing mutant p97. We show that IBMPFD mutations induce conformational changes in the p97 N domain, the main binding site for regulatory co‐factors. Consistently, mutant p97 proteins exhibit strongly altered co‐factor interactions. Specifically, binding of the ubiquitin ligase E4B is reduced, whereas binding of ataxin 3 is enhanced, thus resembling the accumulation of mutant ataxin 3 on p97 in spinocerebellar ataxia type 3. Our results suggest that imbalanced co‐factor binding to p97 is a key pathological feature of IBMPFD and potentially of other proteinopathies involving p97.     

Chromosomal localization of the human proliferating cell nuclear antigen (PCNA) gene to or close to 20p12 by in situ hybridization.

The proliferating cell nuclear antigen (PCNA/cyclin) is described as a nuclear protein and plays a key role in the initiation of cellular DNA synthesis and regulation of cell cycle progression. Using in situ hybridization, the human PCNA gene is localized on chromosome 20 at or close to 20p12.