Post-translation modification of the fourth component of complement. Effect of tunicamycin and amino acid analogs on the formation of the internal thiol ester and disulfide bonds

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The appearance of a functional thiol ester within murine pro-C4 (the intracellular precursor of C4) has been studied. This was assessed by testing the ability of pro-C4 molecules to undergo denaturation-dependent autolytic cleavage. In pulse-chase experiments, [35S]methionine-labeled pro-C4 does not autolyze until ~20 min after synthesis by peritoneal macrophages. When intact (not autolyzed) pro-C4 was examined by nonreducing gel electrophoresis, an increase in its apparent M(r) was seen, with a time course similar to that for autolysis. Both the capacity to undergo autolytic cleavage and the M(r) increase were inhibited by cell culture in the presence of the antibiotic tunicamycin or the threonine analog β-hydroxynorvaline, both of which inhibit glycosylation. Upon isolation from tunicamycin- or hydroxynorvaline-treated cells, pro-C4 associates with other cell constituents, probably via disulfide bonds. This phenomenon is not seen with the mature (high M(r)) form of pro-C4 control cultures, and can be prevented if the cells are lysed in the presence of a sulfhydryl reagent such as iodoacetamide. These data suggest that the post-translational modification of pro-C4 includes the acquisition of a disulfidestablized conformation with a greater apparent M(r). This conformation, along with an intact thiol ester, is necessary for autolytic cleavage to occur.

Original languageEnglish (US)
Pages (from-to)14490-14495
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number23
StatePublished - 1983

Fingerprint

Tunicamycin
Sulfhydryl Compounds
Disulfides
Conformations
Esters
Glycosylation
Iodoacetamide
Amino Acids
Sulfhydryl Reagents
Denaturation
Macrophages
Threonine
Electrophoresis
Cell culture
Methionine
Autolysis
Gels
Cells
Peritoneal Macrophages
Post Translational Protein Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{9f57a993f1934fb9bca96fa736691f9b,
title = "Post-translation modification of the fourth component of complement. Effect of tunicamycin and amino acid analogs on the formation of the internal thiol ester and disulfide bonds",
abstract = "The appearance of a functional thiol ester within murine pro-C4 (the intracellular precursor of C4) has been studied. This was assessed by testing the ability of pro-C4 molecules to undergo denaturation-dependent autolytic cleavage. In pulse-chase experiments, [35S]methionine-labeled pro-C4 does not autolyze until ~20 min after synthesis by peritoneal macrophages. When intact (not autolyzed) pro-C4 was examined by nonreducing gel electrophoresis, an increase in its apparent M(r) was seen, with a time course similar to that for autolysis. Both the capacity to undergo autolytic cleavage and the M(r) increase were inhibited by cell culture in the presence of the antibiotic tunicamycin or the threonine analog β-hydroxynorvaline, both of which inhibit glycosylation. Upon isolation from tunicamycin- or hydroxynorvaline-treated cells, pro-C4 associates with other cell constituents, probably via disulfide bonds. This phenomenon is not seen with the mature (high M(r)) form of pro-C4 control cultures, and can be prevented if the cells are lysed in the presence of a sulfhydryl reagent such as iodoacetamide. These data suggest that the post-translational modification of pro-C4 includes the acquisition of a disulfidestablized conformation with a greater apparent M(r). This conformation, along with an intact thiol ester, is necessary for autolytic cleavage to occur.",
author = "Karp, {D. R.}",
year = "1983",
language = "English (US)",
volume = "258",
pages = "14490--14495",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "23",

}

TY - JOUR

T1 - Post-translation modification of the fourth component of complement. Effect of tunicamycin and amino acid analogs on the formation of the internal thiol ester and disulfide bonds

AU - Karp, D. R.

PY - 1983

Y1 - 1983

N2 - The appearance of a functional thiol ester within murine pro-C4 (the intracellular precursor of C4) has been studied. This was assessed by testing the ability of pro-C4 molecules to undergo denaturation-dependent autolytic cleavage. In pulse-chase experiments, [35S]methionine-labeled pro-C4 does not autolyze until ~20 min after synthesis by peritoneal macrophages. When intact (not autolyzed) pro-C4 was examined by nonreducing gel electrophoresis, an increase in its apparent M(r) was seen, with a time course similar to that for autolysis. Both the capacity to undergo autolytic cleavage and the M(r) increase were inhibited by cell culture in the presence of the antibiotic tunicamycin or the threonine analog β-hydroxynorvaline, both of which inhibit glycosylation. Upon isolation from tunicamycin- or hydroxynorvaline-treated cells, pro-C4 associates with other cell constituents, probably via disulfide bonds. This phenomenon is not seen with the mature (high M(r)) form of pro-C4 control cultures, and can be prevented if the cells are lysed in the presence of a sulfhydryl reagent such as iodoacetamide. These data suggest that the post-translational modification of pro-C4 includes the acquisition of a disulfidestablized conformation with a greater apparent M(r). This conformation, along with an intact thiol ester, is necessary for autolytic cleavage to occur.

AB - The appearance of a functional thiol ester within murine pro-C4 (the intracellular precursor of C4) has been studied. This was assessed by testing the ability of pro-C4 molecules to undergo denaturation-dependent autolytic cleavage. In pulse-chase experiments, [35S]methionine-labeled pro-C4 does not autolyze until ~20 min after synthesis by peritoneal macrophages. When intact (not autolyzed) pro-C4 was examined by nonreducing gel electrophoresis, an increase in its apparent M(r) was seen, with a time course similar to that for autolysis. Both the capacity to undergo autolytic cleavage and the M(r) increase were inhibited by cell culture in the presence of the antibiotic tunicamycin or the threonine analog β-hydroxynorvaline, both of which inhibit glycosylation. Upon isolation from tunicamycin- or hydroxynorvaline-treated cells, pro-C4 associates with other cell constituents, probably via disulfide bonds. This phenomenon is not seen with the mature (high M(r)) form of pro-C4 control cultures, and can be prevented if the cells are lysed in the presence of a sulfhydryl reagent such as iodoacetamide. These data suggest that the post-translational modification of pro-C4 includes the acquisition of a disulfidestablized conformation with a greater apparent M(r). This conformation, along with an intact thiol ester, is necessary for autolytic cleavage to occur.

UR - http://www.scopus.com/inward/record.url?scp=0020973466&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020973466&partnerID=8YFLogxK

M3 - Article

C2 - 6643498

AN - SCOPUS:0020973466

VL - 258

SP - 14490

EP - 14495

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 23

ER -