Biochemical properties of lens-specific calpain Lp85

M. Shih, Hong Ma, E. Nakajima, Larry David, M. Azuma, Thomas (Tom) Shearer

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K50%act=20 μM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on αA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Original languageEnglish (US)
Pages (from-to)146-152
Number of pages7
JournalExperimental Eye Research
Volume82
Issue number1
DOIs
StatePublished - Jan 2006

Fingerprint

Lenses
Crystallins
Calpain
Calcium
Hydrolysis
Amino Acids
Cysteine Proteases
Baculoviridae
Caseins
m-calpain
calpain Lp82
Affinity Chromatography
Immunoblotting
Cataract
Polyacrylamide Gel Electrophoresis
Rodentia
Mass Spectrometry
Peptide Hydrolases
Fluorescence
Peptides

Keywords

  • Calpain
  • Cataract
  • Lens
  • Lp82
  • Lp85
  • m-calpain

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Biochemical properties of lens-specific calpain Lp85. / Shih, M.; Ma, Hong; Nakajima, E.; David, Larry; Azuma, M.; Shearer, Thomas (Tom).

In: Experimental Eye Research, Vol. 82, No. 1, 01.2006, p. 146-152.

Research output: Contribution to journalArticle

Shih, M. ; Ma, Hong ; Nakajima, E. ; David, Larry ; Azuma, M. ; Shearer, Thomas (Tom). / Biochemical properties of lens-specific calpain Lp85. In: Experimental Eye Research. 2006 ; Vol. 82, No. 1. pp. 146-152.
@article{e38512ff2c484578b165f69ac697a738,
title = "Biochemical properties of lens-specific calpain Lp85",
abstract = "Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K50{\%}act=20 μM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on αA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.",
keywords = "Calpain, Cataract, Lens, Lp82, Lp85, m-calpain",
author = "M. Shih and Hong Ma and E. Nakajima and Larry David and M. Azuma and Shearer, {Thomas (Tom)}",
year = "2006",
month = "1",
doi = "10.1016/j.exer.2005.06.011",
language = "English (US)",
volume = "82",
pages = "146--152",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Biochemical properties of lens-specific calpain Lp85

AU - Shih, M.

AU - Ma, Hong

AU - Nakajima, E.

AU - David, Larry

AU - Azuma, M.

AU - Shearer, Thomas (Tom)

PY - 2006/1

Y1 - 2006/1

N2 - Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K50%act=20 μM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on αA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

AB - Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K50%act=20 μM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on αA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

KW - Calpain

KW - Cataract

KW - Lens

KW - Lp82

KW - Lp85

KW - m-calpain

UR - http://www.scopus.com/inward/record.url?scp=30344481119&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=30344481119&partnerID=8YFLogxK

U2 - 10.1016/j.exer.2005.06.011

DO - 10.1016/j.exer.2005.06.011

M3 - Article

VL - 82

SP - 146

EP - 152

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

IS - 1

ER -