Facile cloning and sequencing of S-crystallin genes from octopus lenses based on polymerase chain reaction.
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Cloned OCTS1 cDNA from octopus lens poly(A)+RNA using PCR
"S-crystallin is a major lens protein present in the octopus and squid of Cephalopods. To facilitate the cloning of the protein, cDNA was constructed from the poly(A)+RNA of octopus lenses, and amplification by polymerase chain reaction (PCR) was carried out."
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S-crystallins exist as a multigene family with 37-44% nucleotide similarity and 23-30% amino acid similarity to mammalian GSTs
"Sequencing two of 15 positive clones obtained shows 37-44% similarity in nucleotide and 23-30% similarity in amino acid sequences as compared with mammalian glutathione S-transferases (GST), revealing that S-crystallins exist as a multigene family and probably derived from GST by gene duplication and subsequent mutational base replacements."
Octopus S-crystallins with endogenous glutathione S-transferase (GST) activity: sequence comparison and evolutionary relationships with authentic GST enzymes.
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Three distinct S-crystallin isoforms identified with 61-64% nucleotide identity
"Sequencing of 10 of 15 positive clones coding for this crystallin revealed three distinct S-crystallin isoforms with 61-64% identity in nucleotide sequences and 42-58% similarity in amino acid sequences when compared with homologous crystallins in squid lenses."
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Recombinant octopus S-crystallin has much lower GST activity than authentic GSTs
"We found that the expressed octopus S-crystallin possessed much lower GST activity than the authentic GSTs from other tissues."
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S-crystallins are structurally related to Alpha-class GSTs and derived from ancestral GST
"Sequence comparison and construction of phylogenetic trees for S-crystallins from squid and octopus lenses and various classes of GSTs revealed that S-crystallins represent a multigene family which is structurally related to Alpha-class GSTs and probably derived from the ancestral GST by gene duplication and subsequent multiple mutational substitutions."
Glutathione S-transferase and S-crystallins of cephalopods: evolution from active enzyme to lens-refractive proteins.
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At least 24 different S-crystallins in squid Loligo opalescens, 46-99% identical
"Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46-99% identical to each other by amino acid sequence in the squid Loligo opalescens."
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Long-loop S-crystallins have no GST activity; short-loop retain some
"Squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate."
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GST activity lost by gradual sequence drift plus insertion of central peptide by exon shuffling
"These
data indicate that the S-crystallins consist of a family of enzymatically
inactive proteins (when using CDNB as a substrate) which is considerably larger
than previously believed and that GST activity was lost by gradual drift in
sequence as well as by insertion of an extra peptide by exon shuffling."
Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.
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OctS4 Q108F mutant structure in complex with GSH at 2.35 angstrom resolution; homolog evidence for OCTS1
"We determined the crystal structure of the S-crystallin Q108F mutant at 2.35 Å resolution"
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Wild-type OctS4 has markedly lower GST kcat and catalytic efficiency than authentic octopus GST-sigma
"The GST catalytic activity (kcat) of the wild-type S-crystallin is 0.24 s−1, which is about the same to that of the S-crystallins purified from octopus lens18 but only ~1/700, of that of GST-σ (it will be ~1/6000 if compared their catalytic efficiency by kcat/Km,CDNB) (Table 1)."
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OctS4 has a 43-fold lower apparent Km for GSH than GST-sigma; this is not a directly measured binding Kd
"the apparent binding affinity of GSH with S-crystallin is significantly tighter than that with GST-σ, with a 43-fold decrease in Km,GSH."
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GSH increases OctS4 thermal stability; homolog evidence for a stabilization mechanism
"In the presence of GSH, the melting temperature (Tm) of S-crystallin was higher by 7 °C than that of the protein in the absence of GSH"
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The OctS4 quadruple mutant shows a 518-fold increase in catalytic efficiency toward CDNB
"Furthermore, the quadruple mutant of S-crystallin, L100F/D101N/M104V/Q108F, had the lowest Km,CDNB (0.18 mM) and the highest Km,GSH. Although there was only a 23-fold increase in activity for some unknown reason, the catalytic efficiency (kcat/Km,CDNB) has a 518-fold increase (Table 1)."
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Tradeoff between enzyme activity and protein stability via GSH binding drove evolution
"a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution"
Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.
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Squid lens crystallins confirmed as a family of GST-like proteins by peptide sequencing
"Here we confirm by peptide sequencing that the crystallins of the
lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like
proteins."
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Squid lens extracts showed 400 times less GST activity than liver
"Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity."
Evolution of graded refractive index in squid lenses.
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S-crystallins differentially expressed in a radial gradient correlating with refractive index
"S-crystallins are differentially expressed in a radial gradient, suggesting a role in refractive index. This gradient in S-crystallin expression is correlated with their evolutionary history and biochemistry."
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S-crystallins have been under positive selection for protein stabilization
"S-crystallins have been under positive selection. This selection appears to have resulted in stabilization of derived S-crystallins via mutations in the dimer interface and extended electrostatic fields."
[The evolutionary kinship of the crystallins of cephalopods and vertebrates with heat-shock proteins and stress-induced proteins].
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S-crystallins show no enzymatic activity despite homology with squid GST
"S-crystallins of the squid and the octopus are related, but not identical to glutathione S-transferases. S-crystallins show no enzymatic activity, though they have 42-44% homology with a squid glutathione-S-transferase."
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Vertebrates and cephalopods converged on same strategy of enzyme co-option for lens function
"In the course of evolution vertebrates and cephalopods used the same strategy of recruitment of ancestral proteins (primarily enzymes, whose activity is related with stress and detoxification) for structural functions in the lens."
Eye patches: Protein assembly of index-gradient squid lenses.
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S-crystallins form colloidal gels at all radial positions in the squid lens via patchy colloidal self-assembly
"patchy colloidal physics resulted from an evolutionary radiation of globular S-crystallin proteins"
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Disordered loops protruding from the S-crystallin surface (including the long-loop insertion) serve as low-valence linkers for self-assembly into volumetric materials
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Peripheral lens regions with low particle valence form stable gels at low density, while central regions with higher valence gel at higher densities, creating the refractive index gradient
Molecular basis for the polymerization of octopus lens S-crystallin.
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Octopus S-crystallin aggregates more easily than sigma-GST in the presence of denaturants
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Proposed molecular model involves side-by-side associations of Lys-208 with a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, Asp-180), potentially forming a liquid crystal structure in the lens
Homology modeling of cephalopod lens S-crystallin: a natural mutant of sigma-class glutathione transferase with diminished endogenous activity.
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Homology model revealed that S-crystallin active center is more buried after dimerization than in GST-sigma
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Mutation of Asn99 (GST) to Asp101 (S-crystallin) alters the electrostatic environment at the active site, contributing to loss of catalytic activity
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The closed conformation explains the failure of S-crystallin to bind immobilized glutathione in affinity chromatography
Kinetic characterization of the endogenous glutathione transferase activity of octopus lens S-crystallin.
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S-crystallin kinetics conform to a steady-state random Bi-Bi mechanism similar to authentic GSTs
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Tyr7 interacts with bound GSH to lower the pKa of the sulfhydryl group to 6.82-6.85, but overall catalytic efficiency is drastically reduced
Membrane structures and functional correlates in the bi-segmented eye lens of the cephalopod.
TreeGrafter-generated GO annotations
Blinded OpenScientist function-assignment report (TreeGrafter audit)
Existing falcon OCTS1 function-hypothesis report