用利用基因選殖,表現和定點突變法對具分子保護者活性之A-水晶體蛋白機制研究(3/3)
Date Issued
2003-10-31
Date
2003-10-31
Author(s)
邱式鴻
DOI
912311B002035
Abstract
α-Crystallin, a major protein of all
vertebrate lenses, consisting of two
subunits αA and αB, forms polymeric
aggregates with an average molecular
mass of about 800 kDa. In this study, we
have employed various physical
methods to study aggregate sizes and
conformational properties of porcine
α-crystallin and homomultimeric
aggregates of its purified αA, αB
subunits and cloned recombinant αB
subunit. From far- and near-UV CD
spectra, native α-, αA-, αB- and
recombinant αB-crystallins from
porcine lenses all show similar β-sheet
conformation to that from bovine and
human lenses. By means of gel-filtration
chromatography and dynamic light
scattering, we have found that the
molecular sizes of all four crystallin
aggregates are polydispersedly
distributed in the following order of
aggregate sizes, i.e., native α > αA > αB
≈ recombinant αB. In contrast, the
molecular weights as determined from
sedimentation velocity and equilibrium
studies appear to indicate that the
molecular size of native or recombinant
αB-crystallin is somewhat greater than
αA-crystallin. To further investigate the
structural and functional relationship,
we have also compared the chaperone
activities of all four α-crystallin
aggregates at different temperatures.
From the results of chaperone-activity
assays, ANS (8-anilo-1-naphthalene
sulfonate) binding and thermal stability
studies, there appeared to be at least two
factors playing major roles in the
chaperone-like activity of these lens
proteins: one is the hydrophobicity of
exposed protein surface and the other is
the structural stability associated with
each protein. We showed that
αA-crystallin is a better chaperone to
protect γ-crystallin against
UV-irradiation than αB-crystallin, in
contrast to the observation that αB is
generally a better chaperoning protein
than αA for enzyme protective assays at
physiological temperatures.
We have also analyzed, expressed
and characterized the catfish αB-crystallin, which presents itself as a
major structural component in eye lenses
and as a small heat shock protein in
other tissues of most mammals.
Catfishes which reside generally in
streams, ponds, ditches and reservoirs,
always in the dark or underground
environment, are good examples of
nocturnal scavengers with atrophied
eyes. Sequence comparison with
homologues of distantly related taxa has
revealed conservation of intron splicing
sites and coding regions; however the
two intron sequences, 5’ and 3’
untranslated regions of catfish
αB-crystallin are shorter than those
reported for other vertebrates. The
deduced amino-acid sequence of catfish
αB-crystallin gene was found to lack a
segment of three amino acids (SPF, i.e.
Ser-Pro-Phe) in the N-terminal region,
which were conserved in all of its
homologues characterized from other
vertebrate classes. The phylogenetic
analysis based on protein sequences
indicated that the molecular evolution of
αB-crystallins of dogfish, zebrafish and
catfish followed three independent
evolutionary routes, distinct from
αB-crystallins of other vertebrate
species. The wild type and insertion
mutant (+SPF mutant) of catfish
αB-crystallins were expressed and
characterized. The most striking feature
was that the midpoint for aggregation
(Ta) of the purified recombinant catfish
αB-crystallin was 15°C higher than that
of porcine one, whereas Ta of +SPF
mutant was slightly lower than that of
wild type. The molecular mass of +SPF
mutant as determined by analytical gel
filtration is higher than that of the wild
type (~440 kDa) and similar to porcine
one (~600 kDa). The relative order of
chaperone activity was found to be as
follows: catfish wild type > catfish +SPF
mutant > porcine wild type
αB-crystallin for enzyme protection
assay at 60°C and insulin reduction
assay at 37°C. The surface
hydrophobicity as determined by
fluorescent dye-binding analysis for the
wild type αB-crystallins of catfish is
similar to that of porcine but higher than
that of catfish +SPF mutant. Therefore
both aggregate size and surface
hydrophobicity of crystallin may play
some role in the chaperone-like function of αB-crystallin.
Subjects
水晶體蛋白
分子保護者
抗熱「保護者」活性
老年性白內
障
障
熱休克蛋白
Publisher
臺北市：國立臺灣大學生化科學研究所
Type
report
File(s)![Thumbnail Image]()
Loading...
Name
912311B002035.pdf
Size
40.13 KB
Format
Adobe PDF
Checksum
(MD5):7b81ea461bcafec596316a3b2c8dd513