Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

Suhng Wook Kim, Mary Ann Quinn-Allen, J. Terese Camp, Sandra Macedo-Ribeiro, Pablo Fuentes-Prior, Wolfram Bode, William H. Kane

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg2074, Asp2098, Arg2171, Arg2174, and Glu2189 are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg2074, Asp2098, Arg2171, and Arg2174) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys2060 through Glu2069 in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His2128 through Lys2137. The PS-binding site in the factor V C2-domain includes amino acid residues Trp2063 and Trp2064. This site overlaps with the epitope for monoclonal antibody HV- 1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

Original languageEnglish
Pages (from-to)1951-1958
Number of pages8
JournalBiochemistry
Volume39
Issue number8
DOIs
Publication statusPublished - 2000 Feb 29
Externally publishedYes

Fingerprint

Mutagenesis
Factor V
Human engineering
Alanine
Scanning
Amino Acids
Epitopes
Phosphatidylserines
Monoclonal Antibodies
Membranes
Molecular interactions
C2 Domains
COS Cells
Factor VIII
Hemophilia A
Structural integrity
Point Mutation
Phospholipids
Enzyme-Linked Immunosorbent Assay
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kim, S. W., Quinn-Allen, M. A., Camp, J. T., Macedo-Ribeiro, S., Fuentes-Prior, P., Bode, W., & Kane, W. H. (2000). Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis. Biochemistry, 39(8), 1951-1958. https://doi.org/10.1021/bi992256r

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis. / Kim, Suhng Wook; Quinn-Allen, Mary Ann; Camp, J. Terese; Macedo-Ribeiro, Sandra; Fuentes-Prior, Pablo; Bode, Wolfram; Kane, William H.

In: Biochemistry, Vol. 39, No. 8, 29.02.2000, p. 1951-1958.

Research output: Contribution to journalArticle

Kim, SW, Quinn-Allen, MA, Camp, JT, Macedo-Ribeiro, S, Fuentes-Prior, P, Bode, W & Kane, WH 2000, 'Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis', Biochemistry, vol. 39, no. 8, pp. 1951-1958. https://doi.org/10.1021/bi992256r
Kim, Suhng Wook ; Quinn-Allen, Mary Ann ; Camp, J. Terese ; Macedo-Ribeiro, Sandra ; Fuentes-Prior, Pablo ; Bode, Wolfram ; Kane, William H. / Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis. In: Biochemistry. 2000 ; Vol. 39, No. 8. pp. 1951-1958.
@article{d241380f2bee483e909c1a75529fd34f,
title = "Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis",
abstract = "We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg2074, Asp2098, Arg2171, Arg2174, and Glu2189 are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg2074, Asp2098, Arg2171, and Arg2174) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys2060 through Glu2069 in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His2128 through Lys2137. The PS-binding site in the factor V C2-domain includes amino acid residues Trp2063 and Trp2064. This site overlaps with the epitope for monoclonal antibody HV- 1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.",
author = "Kim, {Suhng Wook} and Quinn-Allen, {Mary Ann} and Camp, {J. Terese} and Sandra Macedo-Ribeiro and Pablo Fuentes-Prior and Wolfram Bode and Kane, {William H.}",
year = "2000",
month = "2",
day = "29",
doi = "10.1021/bi992256r",
language = "English",
volume = "39",
pages = "1951--1958",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

AU - Kim, Suhng Wook

AU - Quinn-Allen, Mary Ann

AU - Camp, J. Terese

AU - Macedo-Ribeiro, Sandra

AU - Fuentes-Prior, Pablo

AU - Bode, Wolfram

AU - Kane, William H.

PY - 2000/2/29

Y1 - 2000/2/29

N2 - We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg2074, Asp2098, Arg2171, Arg2174, and Glu2189 are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg2074, Asp2098, Arg2171, and Arg2174) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys2060 through Glu2069 in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His2128 through Lys2137. The PS-binding site in the factor V C2-domain includes amino acid residues Trp2063 and Trp2064. This site overlaps with the epitope for monoclonal antibody HV- 1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

AB - We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg2074, Asp2098, Arg2171, Arg2174, and Glu2189 are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg2074, Asp2098, Arg2171, and Arg2174) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys2060 through Glu2069 in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His2128 through Lys2137. The PS-binding site in the factor V C2-domain includes amino acid residues Trp2063 and Trp2064. This site overlaps with the epitope for monoclonal antibody HV- 1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

UR - http://www.scopus.com/inward/record.url?scp=0001252591&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0001252591&partnerID=8YFLogxK

U2 - 10.1021/bi992256r

DO - 10.1021/bi992256r

M3 - Article

C2 - 10684644

AN - SCOPUS:0001252591

VL - 39

SP - 1951

EP - 1958

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 8

ER -