Pigmentation-Associated Glycoprotein of Human Melanomas and Melanocytes: Definition with a Mouse Monoclonal Antibody

Abstract

Pigmented melanoma cells and cultured melanocytes express a differentiation-related glycoprotein designated as pigmentation-associated antigen (PAA) of Mr 70,000-80,000. As described previously, PAA was initially defined by reactivity with antibodies in the serum of a patient with melanoma. Here we describe the production and characterization of a mouse monoclonal antibody to PAA. This antibody (TA99, an IgG2a) was shown by sequential immunoprecipitation experiments to react with the same component as the human antibody. Ab TA99 immunoprecipitated PAA from lysates of cells radiolabeled with [35S]methionine, [3H]glucosamine, [3H]fucose, and [3H]mannose as well as 125I. Using Ab TA99, the distribution of PAA was examined in frozen sections of 19 normal tissues and quantitatively in 68 tissue culture cell lines. In frozen sections, only melanin-containing cells were positive, including epithelial cells in the basal layer of the epidermis, in which pigment originates from melanocytes by transfer of melanosomes, and pigmented cells of the eye. In tissue culture cell lines, only pigmented melanoma cells were positive. PAA is an intracellular antigen, with a distribution very similar to that of melanosomes. This evidence confirms the close association of PAA with melanin production, and suggests that PAA may be a melanosome component. PAA was shown to be different from tyrosinase, the enzyme involved in melanin synthesis, but it was found to be identical to the previously recognized glycoprotein, gp75, characteristic of pigmented melanomas and melanocytes.

The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product.

“…Both mouse mAb TA99 and serum from melanoma patient AU immunoprecipitate a 75-kD antigen, and mAb TA99 preclears the gp75 antigen precipitated by AU serum (9) . Since mAb TA99 was used to purify gp75 from the melanoma cell line SK-MEL-19, it was important to confirm that mAb TA99 detected only the gp75 antigen recognized by AU serum .…”

TRP‐1 expression correlates with eumelanogenesis in human pigment cells in culture

“…Interestingly, the presence of gp75 has been associated with visible pi~en~tion of human melanoma cells [13]. In the present work, we have examined in more detail the relationship between tyrosinase and TRPllBrown mRNA accumulation, tyrosinase activity and the presence of eumelanins or pheomelanins.…”

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma

“…The paraformaldehyde was inactivated with 5 mm NH 4 CL, the cells permeabilized with 0.01% saponin with 1% bovine serum albumin in phosphate-buffered saline (pH 7.4), stained with the TA99 anti-gp75 monoclonal antibody (1:200 dilution), and counterstained with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody. 81 Representative fields were imaged with a Nikon Microphot SA, equipped with a ×40, 0.7 NA objective lens, Nikon FITC epifluorescence filter cube, and differential interference contrast optics (Nikon, Melville, NY, USA). Images were collected with a cooled chargecoupled device camera (Princeton Instruments, Trenton, NJ, USA).…”