Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.