Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon

An antisense study

Mark E. Rothenberg, Carmen D. Eilertson, Kathy Klein, Robert Mackin, Bryan D. Noe

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, αTC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 ± 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 ± 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Original languageEnglish
Pages (from-to)331-341
Number of pages11
JournalMolecular Endocrinology
Volume10
Issue number4
DOIs
StatePublished - 1996

Fingerprint

Proglucagon
Proprotein Convertases
Glicentin
Peptides
Transfection
Peptide Mapping
Cytotoxic T-Lymphocytes
Glucagon
Tryptophan
Radioactivity
Complementary DNA
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon : An antisense study. / Rothenberg, Mark E.; Eilertson, Carmen D.; Klein, Kathy; Mackin, Robert; Noe, Bryan D.

In: Molecular Endocrinology, Vol. 10, No. 4, 1996, p. 331-341.

Research output: Contribution to journalArticle

Rothenberg, Mark E. ; Eilertson, Carmen D. ; Klein, Kathy ; Mackin, Robert ; Noe, Bryan D. / Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon : An antisense study. In: Molecular Endocrinology. 1996 ; Vol. 10, No. 4. pp. 331-341.
@article{6166c998353c467d929bf9c3968292c9,
title = "Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: An antisense study",
abstract = "To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, αTC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 ± 7.9{\%} but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 ± 11.7{\%} without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.",
author = "Rothenberg, {Mark E.} and Eilertson, {Carmen D.} and Kathy Klein and Robert Mackin and Noe, {Bryan D.}",
year = "1996",
doi = "10.1210/me.10.4.331",
language = "English",
volume = "10",
pages = "331--341",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "4",

}

TY - JOUR

T1 - Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon

T2 - An antisense study

AU - Rothenberg, Mark E.

AU - Eilertson, Carmen D.

AU - Klein, Kathy

AU - Mackin, Robert

AU - Noe, Bryan D.

PY - 1996

Y1 - 1996

N2 - To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, αTC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 ± 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 ± 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

AB - To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, αTC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 ± 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 ± 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

UR - http://www.scopus.com/inward/record.url?scp=0029925365&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029925365&partnerID=8YFLogxK

U2 - 10.1210/me.10.4.331

DO - 10.1210/me.10.4.331

M3 - Article

VL - 10

SP - 331

EP - 341

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 4

ER -