Characterisation and purification of an aggrecanase made by injured synovium
File(s)
Author(s)
Rana, Faisal
Type
Thesis
Abstract
Freshly dissected porcine synovial tissue in culture produces an enzymatic activity that
cleaves cartilage aggrecan generating ARGS- and AGEG- bearing neo-epitope fragments.
The aggrecanolytic activity was abolished when synovial tissue was cultured in the presence
of cycloheximide. The enzyme(s) were sensitive to N-terminal inhibitory domain of tissue
inhibitor of matrix metalloproteinase (N-TIMP-3) and general matrix metalloproteinase
inhibitor (GM6001) suggesting they may belong to a disintegrin and metalloproteinase
(ADAM) or ADAM with thrombospondin motifs (ADAMTS) family of enzymes. Cation
exchange chromatography was used to partially purify aggrecanase(s) from synovial tissue
culture medium (SYCM). Two active species have been separated from the partially purified
material using size-exclusion chromatography. The smaller species had a molecular weight
of 35-40 kDa while the larger enzyme had an apparent molecular weight greater than 2000
kDa. Low density lipoprotein receptor-related protein (LRP1) didn’t appear to be involved in
the formation of higher molecular weight complex. The smaller species was further
chromatographed on a SMART mono Q column. The sequential chromatography gave
approximately 400-fold enrichment of the enzyme. The concentration of the enzyme was
estimated by titration with recombinant N-TIMP-3, which was expressed and purified from
E.coli. The N-TIMP-3 was electrostatically coupled to Ni2+ agarose beads. The beads were
then used to affinity purify the enzyme from mono Q fractions. The affinity-purified material
was electrophoresed and protein bands were selected for mass spectrometry. No ADAMTS
enzyme was identified in the candidate bands. Further improvements will be made to the
purification procedure to identify the synovial aggrecanase.
cleaves cartilage aggrecan generating ARGS- and AGEG- bearing neo-epitope fragments.
The aggrecanolytic activity was abolished when synovial tissue was cultured in the presence
of cycloheximide. The enzyme(s) were sensitive to N-terminal inhibitory domain of tissue
inhibitor of matrix metalloproteinase (N-TIMP-3) and general matrix metalloproteinase
inhibitor (GM6001) suggesting they may belong to a disintegrin and metalloproteinase
(ADAM) or ADAM with thrombospondin motifs (ADAMTS) family of enzymes. Cation
exchange chromatography was used to partially purify aggrecanase(s) from synovial tissue
culture medium (SYCM). Two active species have been separated from the partially purified
material using size-exclusion chromatography. The smaller species had a molecular weight
of 35-40 kDa while the larger enzyme had an apparent molecular weight greater than 2000
kDa. Low density lipoprotein receptor-related protein (LRP1) didn’t appear to be involved in
the formation of higher molecular weight complex. The smaller species was further
chromatographed on a SMART mono Q column. The sequential chromatography gave
approximately 400-fold enrichment of the enzyme. The concentration of the enzyme was
estimated by titration with recombinant N-TIMP-3, which was expressed and purified from
E.coli. The N-TIMP-3 was electrostatically coupled to Ni2+ agarose beads. The beads were
then used to affinity purify the enzyme from mono Q fractions. The affinity-purified material
was electrophoresed and protein bands were selected for mass spectrometry. No ADAMTS
enzyme was identified in the candidate bands. Further improvements will be made to the
purification procedure to identify the synovial aggrecanase.
Version
Open Access
Date Issued
2013-09
Date Awarded
2014-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Saklatvala, Jeremy
Nagase, Hideaki
Sponsor
Kennedy Trust for Rheumatology Research
Grant Number
AZRXGG0
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)