Localization of sphingomyelinase in lesional skin of atopic dermatitis patients.
Because it has been suggested that the majority of the activity hydrolysing [N-methyl-14C] sphingomyelin is due to sphingomyelin acylase in the lesional skins of atopic dermatitis (AD), in this study we used immunologic techniques to localize and quantitate sphingomyelinase in AD lesional and normal skin. A polyclonal antibody raised against a synthetic polypeptide corresponding to a portion of the amino acid sequence deduced from the cDNA of human acid sphingomyelinase, cross-reacted with a 58 kDa, pI 5.8 human epidermal protein in an immunoblot analysis. The 58 kDa protein-rich fraction, partially purified by immunoprecipitation, converted [N-methyl-14C]-sphingomyelin to 14C-phosphorylcholine and ceramides. The reaction products were immunohistochemically observed in the intercellular domain from the upper spinous cell layer to the upper stratum corneum cell layers in the lesional skin of AD patients. Immunoelectron-microscopically, gold particles appeared to be concentrated in the intercellular domains of the granular-upper stratum corneum cells in the lesional skin of AD patients. The total amount of the 58 kDa protein in a 7 mm2 area of the skin was measured by quantitative immunoblot analysis; and was slightly increased in the lesional skin samples [3.5 +/- 0.3 microg per 7 mm2 (n = 7)], as compared with the nonlesional skin samples of AD patients [2.8 +/- 0.19 microg per 7 mm2 (n = 10)] and with the normal skin samples [2.7 +/- 0.22 microg per 7 mm2 (n = 10)]. This difference (between the lesional skin of AD and the nonlesional skin of AD or the normal control) was significant (nonpaired student's t test, p < 0.05).
Kusuda
S
S
Department of Dermatology, Kinki University School of Medicine, Osaka-Sayama, Osaka, Japan.
Cui
C Y
CY
Takahashi
M
M
Tezuka
T
T
D000906:Antibodies / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D003876:Dermatitis, Atopic / Q000201:enzymology* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D011257:Pregnancy Proteins / Q000276:immunology; D012867:Skin / Q000201:enzymology* / Q000648:ultrastructure; D013108:Sphingomyelin Phosphodiesterase / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D011257:Pregnancy Proteins; D013108:Sphingomyelin Phosphodiesterase