Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII

Mathilde Hunault, Arnaldo A. Arbini, Josephine A. Carew, Flora Peyvandi, Kenneth A. Bauer

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII- Arg100 the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII- Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

Original languageEnglish
Pages (from-to)1237-1244
Number of pages8
JournalBlood
Volume93
Issue number4
Publication statusPublished - Feb 15 1999

Fingerprint

Factor VII
Epidermal Growth Factor
Mutation
Cricetulus
Corrosion inhibitors
Proteolysis
Ovary
Factor VII Deficiency
Cells
Cysteine Proteases
Degradation
COS Cells
Endoplasmic Reticulum
Disulfides
Labeling
Complementary DNA
Molecular Weight
Immunohistochemistry
Proteins
Molecular weight

ASJC Scopus subject areas

  • Hematology

Cite this

Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII. / Hunault, Mathilde; Arbini, Arnaldo A.; Carew, Josephine A.; Peyvandi, Flora; Bauer, Kenneth A.

In: Blood, Vol. 93, No. 4, 15.02.1999, p. 1237-1244.

Research output: Contribution to journalArticle

Hunault, Mathilde ; Arbini, Arnaldo A. ; Carew, Josephine A. ; Peyvandi, Flora ; Bauer, Kenneth A. / Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII. In: Blood. 1999 ; Vol. 93, No. 4. pp. 1237-1244.
@article{722e4cb037194ed9ad52d2b5f468244f,
title = "Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII",
abstract = "We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII- Arg100 the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII- Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.",
author = "Mathilde Hunault and Arbini, {Arnaldo A.} and Carew, {Josephine A.} and Flora Peyvandi and Bauer, {Kenneth A.}",
year = "1999",
month = "2",
day = "15",
language = "English",
volume = "93",
pages = "1237--1244",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "4",

}

TY - JOUR

T1 - Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII

AU - Hunault, Mathilde

AU - Arbini, Arnaldo A.

AU - Carew, Josephine A.

AU - Peyvandi, Flora

AU - Bauer, Kenneth A.

PY - 1999/2/15

Y1 - 1999/2/15

N2 - We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII- Arg100 the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII- Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

AB - We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII- Arg100 the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII- Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

UR - http://www.scopus.com/inward/record.url?scp=0033557948&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033557948&partnerID=8YFLogxK

M3 - Article

VL - 93

SP - 1237

EP - 1244

JO - Blood

JF - Blood

SN - 0006-4971

IS - 4

ER -