Archives of Dermatological Research

, Volume 280, Issue 8, pp 462–468 | Cite as

The 55-kd keratohyalin granule protein has the same epitope as the 43-kd stratum corneum membrane protein: immunofluorescence and immunoblotting studies using a monoclonal antibody to the 55-kd keratohyalin granule protein

  • T. Tezuka
  • M. Takahashi
Original Contributions


A monoclonal antibody (Ted-R-1) to the keratohyalin granules (KHGs) of the newborn rat epidermis was developed by immunizing mice with a pH 4.7 hematoxylin-stainable fraction of the extract in a 50 mM Tris-HCl buffer, pH 7.3, containing 10 μ/ml phenylmethylsulfonyl fluoride (PMSF) from the newborn rat whole epidermis. Using an indirect immunofluorescence technique, Ted-R-1 was located in two places: (a) the KHGs and (b) the cell membrane region of the stratum corneum. Under immunoelectron microscopy, the antigenic materials were located at the periphery of the KHGs. Immunoblotting analyses of the antigenic pH 4.7 fraction, the Tris-HCl extract from the stratum corneum, and the Tris-8M urea extract from freeze-dried whole skin demonstrated that there were 55 kd, 43 kd, and 78 kd polypeptides, respectively, with which Ted-R-1 reacts. No possitive spot was seen in the dermal extract. The major amino acids of the 55 kd protein were glycine (24.4%), serine (17.3%), glutamic acid (14.1%), and alanine (7.7%), while the histidine residue was only 3.1%. On the basis of its amino acid composition, the 55 kd protein is the third component of KHGs — in addition to histidine-rich and cystinerich proteins.

Key words

Hematoxylin stainable protein Keratohyalin granules Membrane lining protein Stratum corneum 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ball RD, Walker GK, Bernstein IA (1979) Histidine-rich proteins as molecular markers of epidermal differentiation. J Biol Chem 253:5861–5868Google Scholar
  2. 2.
    Clezardin P, Hunter NR, McGregor IR, McGregor JL, Peppers DS, Dawes J (1986) Tandem purification of mouse IgM monoclonal antibodies produced in vitro using anion exchange and gel fast protein liquid chromatography. J Chromatogr 358:209–218Google Scholar
  3. 3.
    Dale BA, Lonsdale-Eccles JD, Holbrook KA (1980) Stratum corneum basic protein: an interfilamentous matrix protein of epidermal keratin. In: Bernstein IA, Seiji M (eds) Biochemistry of normal and abnormal epidermal differentiation. Tokyo University Press, Tokyo, pp 311–325Google Scholar
  4. 4.
    Fukuyama K, Epstein WL (1975) A comparative autoradiographic study of keratohyalin granules containing cystine and histidine, J Ultrastruct Res 51:314–325Google Scholar
  5. 5.
    Gershoni JM, Palade GE (1982) Electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem 124:396–405Google Scholar
  6. 6.
    Hattori T, Manabe M, Ogawa H (1981) Characteristics of horny cell membrane isolated from the stratum corneum. Jpn J Dermatol 91:677–680Google Scholar
  7. 7.
    Hurrell JGR (1982) Monoclonal hybridoma antibodies: techniques and applications. CRC Press, Boca Raton, Florida, pp 20–36Google Scholar
  8. 8.
    Jackle H (1979) Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis. Anal Biochem 98:81–84Google Scholar
  9. 9.
    Jessen H, Peters PD, Hall TA (1974) Sulphur in different types of keratohyalin granules: a quantitative assay by X-ray microanalysis. J Cell Sci 15:359–377Google Scholar
  10. 10.
    Kawamura A Jr (1977) Fluorescent antibody techniques and their applications, 2nd edn. University of Tokyo Press, Tokyo, pp 77–94Google Scholar
  11. 11.
    Kristiansen T (1978) Affinity chromatography. Matrix-bound antigens and antibodies. In: Hoffman-Osenhof (ed) Affinity chromatography. Pergamon Press; Oxford, pp 191–206Google Scholar
  12. 12.
    Matoltsy AG, Matoltsy MN (1966) The membrane protein of horny cells. J Invest Dermatol 46:127–129Google Scholar
  13. 13.
    Murozuka T, Fukuyama K, Epstein WL (1979) Immunological comparison of histidine-rich protein in keratohyalin granules and cornified cells. Biochim Biophys Acta 579:334–345Google Scholar
  14. 14.
    Sibrack LA, Gray RH, Bernstein IA (1974) Localizaation of the histidine-rich protein in keratohyalin. A morphologic and macromolecular marker in epidermal differentiation. J Invest Dermatol 62:394–405Google Scholar
  15. 15.
    Tezuka T (1975) The extraction and partial purification of the deoxycholate soluble matrix protein from the human plantar horny layer. Acta Derm Venereol (Stockh) 55:401–412Google Scholar
  16. 16.
    Tezuka T (1984) Two cases of hereditary palmoplantar keratoderma. An abnormality of keratohyalin granules and keratin formation. Dermatologica 169:138–145Google Scholar
  17. 17.
    Tezuka T, Freedberg IM (1978) The epidermal structural proteins. III. Isolation and purification of histidine-rich protein of the newborn rat. J Invest Dermatol 63:402–406Google Scholar
  18. 18.
    Tezuka T, Hirai R (1978) The constituents of keratohyalin granules of newborn rat epidermis under an electron microscopy. Acta Derm Venereol (Stockh) 58:285–289Google Scholar
  19. 19.
    Tezuka T, Takahashi M (1986) A novel protein of rat epidermis. Haematoxylin stain-positive protein of keratohyalin granules as the membrane lining protein of the stratum corneum. J Dermatol 13:417–425Google Scholar
  20. 20.
    Tezuka T, Takahashi M (1987) The cystine-rich envelope protein from human epidermal stratum corneum cells. J Invest Dermatol 88:47–51Google Scholar
  21. 21.
    Tezuka T, Takahashi M (1987) Stratum corneum membrane proteins in newborn rat as scavengers of lipid peroxide. In: Hayaishi O, Imamura S, Miyachi Y (eds) The biological role of reactive oxygen species in skin. University of Tokyo Press, Tokyo, pp 125–132Google Scholar
  22. 22.
    Tezuka T, Takahashi M (1987) Human hematoxylin stainable protein of keratohyalin granules origin. I. Extraction and purification. J Invest Dermatol 89:400–404Google Scholar
  23. 23.
    Ugel AR (1969) Keratohyalin, extraction and in vitro aggregation. Science 166:250–251Google Scholar
  24. 24.
    Ugel AR (1971) Studies on isolated aggregating origoribonucleo-proteins of the epidermis with histochemical and ultrastructural characteristics of keratohyalin. J Cell Biol 49:405–422Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • T. Tezuka
    • 1
  • M. Takahashi
    • 1
  1. 1.Department of DermatologyKinki University School of MedicineOsaka-sayama-shi, OsakaJapan

Personalised recommendations