A-431 cells and human keratinocytes synthesize and secrete the third component of complement

Nicole Basset-Séguin, S. Wright Caughman, Kim B. Yancey

Research output: Contribution to journalArticle

52 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)621-625
Number of pages5
JournalJournal of Investigative Dermatology
Volume95
Issue number6
StatePublished - Dec 1990

Fingerprint

Keratinocytes
Zymosan
Hep G2 Cells
Epithelial Cells
Cells
Proteolysis
Complement C3
Biosynthesis
Basement Membrane
Cell culture
Skin
Demonstrations
Cell Line
RNA
Messenger RNA
Peptides
Molecules
Serum
Northern Blotting
Primates

Keywords

  • American Type Culture Collection
  • ATCC
  • basement membrane
  • BM
  • bovine serum albumin
  • BSA
  • DGVB
  • EDTA
  • ethylenediaminetetraacetic acid
  • Hank's balanced salt solution
  • HBSS
  • HK
  • human keratinocytes
  • immunoprecipitation
  • IP
  • low-ionic-strength Veronal-buffered saline with dextrose, gelatin, Ca, and Mg
  • phenylmethylsulfonyl fluoride
  • PMSF
  • TBS
  • TCA
  • trichloroacetic acid
  • Tris buffered saline

ASJC Scopus subject areas

  • Dermatology

Cite this

A-431 cells and human keratinocytes synthesize and secrete the third component of complement. / Basset-Séguin, Nicole; Wright Caughman, S.; Yancey, Kim B.

In: Journal of Investigative Dermatology, Vol. 95, No. 6, 12.1990, p. 621-625.

Research output: Contribution to journalArticle

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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.",
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AB - 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.

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