Identification of a cDNA coding for a Ca(2+)-binding phosphoprotein (p90), calnexin, on melanosomes in normal and malignant human melanocytes.
In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca(2+)-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in lambda Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M(r) 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with beta-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca(2+)-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an antibody against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.
Dakour
J
J
Division of Dermatology and Cutaneous Sciences, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Vinayagamoorthy
T
T
Jimbow
K
K
Chen
H
H
Luo
D
D
Dixon
W
W
Munoz
V
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D037281:Calnexin; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008544:Melanocytes / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics*; D011233:Precipitin Tests; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D010750:Phosphoproteins; D037281:Calnexin