Mutagenesis of the cysteine-rich clip domain in the Drosophila patterning protease, Snake

Sufang Tian, Ellen LeMosy

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.

Original languageEnglish (US)
Pages (from-to)169-174
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume475
Issue number2
DOIs
StatePublished - Jul 15 2008

Fingerprint

Mutagenesis
Snakes
Surgical Instruments
Cysteine
Assays
Peptide Hydrolases
Bearings (structural)
Defects
Enzyme Precursors
Serine Proteases
Alanine
Transfection
Catalyst activity
Proteins
Substitution reactions
Embryonic Structures
Gastrulation
Aptitude
Structural Models
Invertebrates

Keywords

  • Charged-to-alanine mutagenesis
  • Clip domain
  • Dorsoventral polarity
  • Drosophila
  • Serine protease

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Mutagenesis of the cysteine-rich clip domain in the Drosophila patterning protease, Snake. / Tian, Sufang; LeMosy, Ellen.

In: Archives of Biochemistry and Biophysics, Vol. 475, No. 2, 15.07.2008, p. 169-174.

Research output: Contribution to journalArticle

@article{d9391cad742e41fb93f2461805fe7f2a,
title = "Mutagenesis of the cysteine-rich clip domain in the Drosophila patterning protease, Snake",
abstract = "A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.",
keywords = "Charged-to-alanine mutagenesis, Clip domain, Dorsoventral polarity, Drosophila, Serine protease",
author = "Sufang Tian and Ellen LeMosy",
year = "2008",
month = "7",
day = "15",
doi = "10.1016/j.abb.2008.04.030",
language = "English (US)",
volume = "475",
pages = "169--174",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Mutagenesis of the cysteine-rich clip domain in the Drosophila patterning protease, Snake

AU - Tian, Sufang

AU - LeMosy, Ellen

PY - 2008/7/15

Y1 - 2008/7/15

N2 - A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.

AB - A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.

KW - Charged-to-alanine mutagenesis

KW - Clip domain

KW - Dorsoventral polarity

KW - Drosophila

KW - Serine protease

UR - http://www.scopus.com/inward/record.url?scp=44949089390&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=44949089390&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2008.04.030

DO - 10.1016/j.abb.2008.04.030

M3 - Article

VL - 475

SP - 169

EP - 174

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -