On the analysis of the pathophysiology of Chediak-Higashi syndrome. Defects expressed by cultured melanocytes

Abstract

Background: The Chediak-Higashi syndrome (CHS) is a disorder that affects the synthesis and/or maintenance of storage/secretory granules in various types of cells. Lysosomes of leukocytes and fibroblasts, dense bodies of platelets, azurophilic granules of neutrophils and melanosomes of melanocytes are generally larger in size and irregular in morphology, indicating that a common pathway in storage organellogenesis is affected in patients with CHS.

Experimental design: A pure line of melanocytes has been established using a 2 cm2 shave biopsy from a child with CHS. This 4-week-old male patient had oculocutaneous albinism and expressed neutropenia, impaired platelet function, and no natural killer cell activity. The cultured CHS melanocytes were analyzed for cell biological and biochemical aberrancies.

Results: Cultured melanocytes demonstrated some large and/or complexed melanosomes that resembled those observed in melanocytes from ultrastructural sections of the biopsy. Cytoplasmic localization of tyrosinase, tyrosinase-related protein-1 and granulophysin (a 40 kilodalton membrane protein originally identified as a component in dense bodies of platelets) demonstrated a prominent perinuclear accumulation. The basal synthesis of melanin and the activity levels of tyrosine hydroxylase, dihydroxyphenylalanine (DOPA) oxidase, or DOPAchrome tautomerase were comparable to control Caucasian melanocytes in culture. However, melanin synthesis as well as the catalytic activities of tyrosinase were not dramatically upregulated in CHS melanocytes by the addition of isobutyl methylxanthine and cholera toxin in the growth medium when parameters were assayed in cell lysates. In contrast, when assays were performed using live cells, tyrosine hydroxylase demonstrated dramatic upregulation. Medium conditioned by CHS melanocytes demonstrated phenylthiourea-inhibitable tyrosinase activity. Melanocyte lysates and conditioned medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DOPA staining showed an extra, approximately 100 kilodalton soluble protein band with DOPA positivity and tyrosinase immunoreactivity. In addition to tyrosinase, one of three lysosomal enzymes assayed (beta-glucuronidase) was aberrantly secreted into the medium.

Conclusions: These results demonstrate that melanocytes cultured from CHS express a defect in the structure and/or function of the melanosome and abnormal trafficking of some cellular proteins.