This paper is only available as a PDF. To read, Please Download here.
Abstract
Biosynthetic radiolabeling studies demonstrate that A-431 cells, a human epidermoid
carcinoma cell line, and human keratinocytes synthesize and secrete C3 as two disulfidelinked
polypeptide chains of 120 and 75 kD. Moreover, epithelial cell-derived C3 co-migrates
in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line previously
used to elucidate complement component biosynthesis. Pulse-chase studies in A-431
cells demonstrate that epithelial cell-derived C3 is produced as a 195-kD precursor
molecule, pro-C3, which is processed intracellularly by limited proteolysis into 120-
and 75-kD C3 alpha and beta chains. Comparative studies demonstrate that A-431 cellderived
C3 is synthesized, processed, and secreted in parallel but in lower quantity than
that produced by HepG2 cells. Treatment of biosynthetically labeled A-431 cell culture
supernatants with normal human serum and zymosan produces C3 alpha chain cleavage
and specific C3 fragments that are not present in control culture supernatants treated
with heatinactivated human serum and zymosan. Northern blot analysis of total cellular
RNA extracted from A-431 cells, human keratinocytes, and HepG2 cells reveals qualitative
identity of a 5.1-kb C3 mRNA species in these three cell types. Epithelial cell-derived
C3 may play an important role in local inflammatory and immunologic reactions including
such reactions in human skin. Moreover, epithelial cell C3 synthesis may have direct
relevance to the recent demonstration of C3d,g within selected normal primate epithelial
basement membranes, including epidermal basement membrane.
Keywords
- ATCC American Type Culture Collection
- BM basement membrane
- BSA bovine serum albumin
- DGVB++ low-ionic-strength Veronal-buffered saline with dextrose, gelatin, Ca++, and Mg++
- EDTA ethylenediaminetetraacetic acid
- HBSS Hank's balanced salt solution
- HK human keratinocytes
- IP immunoprecipitation
- PMSF phenylmethylsulfonyl fluoride
- TBS Tris buffered saline
- TCA trichloroacetic acid
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Journal of Investigative DermatologyAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- Complement.in: Paul WE Fundamental Immunology. Raven Press, New York1984: 645-668
- Evidence for presence of an internal thiolester bond in third component of human complement.in: 2nd ed. Proc Natl Acad Sci USA. 77. 1980: 5764-5768
- Molecular mechanisms of complement action.in: Stamatoyannopoulous G Neinhuis A Leder P Majerus P The Molecular Basis of Blood Diseases. W.B. Saunders, St. Louis1987: 450-498
- C3d,g is present in normal human epidermal basement membrane.J Immunol. 1988; 141: 1273-1280
- C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast.J Cell Biol. 1986; 103: 1091-1100
- Defective expression of basement membrane-associated C3d,g in papulonodular basal cell carcinomas.J Invest Dermatol. 1989; 92: 734-738
- Biosynthesis and processing of a human precursor complement protein, pro-C3, in a hepatoma-derived cell line.Science. 1982; 215: 399-400
- Expression of c-Myc, c-Myb, c-Erb-G, and c-H-Ras oncogene mRNAs in fibroblasts cultured from psoriatic patients.Dermatologica. 1987; 175: 296-299
- Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia.Cell. 1981; 24: 897-903
- Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (Lond). 1970; 277: 680-685
- Studies of human C5a as a mediator of inflammation in normal human skin.J Clin Invest. 1985; 75: 486-495
- C3 component of complement secreted by established cell lines.Cell. 1978; 15: 375-384
- Isolation of total RNA by extraction with strong denaturants.in: 2nd ed. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989: 718-722
- Human complement component C3: cDNA coding sequence and derived primary structure.in: Edition 3. Proc Natl Acad Sci USA. 82. 1985: 708-712
- Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide.J Clin Invest. 1985; 76: 985-990
- The epidermal basement membrane zone-structure, ontogeny, and role in disease.J Am Acad Dermatol. 1984; 11: 1025-1037
Article info
Publication history
Accepted:
August 16,
1990
Received:
April 26,
1990
Footnotes
☆This work was supported by USUHS Protocol 208400 (KBY), NIH Grant AR 37446 (KBY), and NATO Scientific Affairs Subvention RG0244/88-3/9/90 (NB-S, KBY). The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
Identification
Copyright
© 1990 Published by Elsevier Inc.
