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Examining the influence of ultraviolet C irradiation on recombinant human γD-crystallin.

Wang SS, Wen WS - Mol. Vis. (2010)

Bottom Line: The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride.Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC.The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan. sswang@ntu.edu.tw

ABSTRACT

Purpose: Human γD crystallin is a principal protein component of the human eye lens and associated with the development of juvenile and mature-onset cataracts. Exposure to ultraviolet (UV) light is thought to perturb protein structure and eventually lead to aggregation. This work is aimed at exploring the effects of UV-C irradiation on recombinant human γD-crystallin (HGDC).

Methods: Recombinant HGDC proteins were expressed in E. coli strain BL21(DE3) harboring plasmid pEHisHGDC and purified using chromatographic methods. The proteins were then exposed to UV-C light (λ(max)=254 nm, 15 W) at the intensity of 420, 800, or 1850 μW/cm(2). The UV-C-unexposed, supernatant fraction of UV-C-exposed, and re-dissolved precipitated fraction of UV-C exposed preparations were characterized by SDS-PAGE, turbidity measurement, CD spectroscopy, tryptophan fluorescence spectroscopy, acrylamide fluorescence quenching analysis, and sulfhydryl group measurements.

Results: The turbidity of the HGDC sample solution was found to be positively correlated with HGDC concentration, UV-C irradiation intensity, and UV-C irradiation duration. When exposed to UV-C, HGDC sample solutions became visibly turbid and a noticeable amount of larger protein particle, perceptible to the naked eye, was observed upon prolonged irradiation. The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride. Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC. Through the use of L-cysteine, the measurements of sulfhydryl contents, and the reducing as well as non-reducing SDS-PAGE, our data further suggested that disulfide bond formation and/or cleavage probably play an important role in aggregation and/or precipitation of HGDC elicited by UV-C irradiation.

Conclusions: Our findings highlight the close connections among disulfide bond cleavage and/or formation, intermolecular interactions, and the resultant formation of aggregates of HGDC induced by UV-C irradiation. The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.

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Related in: MedlinePlus

Non-reducing (without β-mercaptoethanol) SDS–PAGE analysis of UV-C irradiated HGDC samples (1 mg/ml) at different exposure times (M: protein marker, Lane 1: 0 min, Lane 2: 10 min, Lane 3: 20 min, Lane 4: 30 min, Lane 5: 40 min, Lane 6: 50 min, Lane 7: 60 min). The dose of UV-C irradiation used was 1,850 μW/cm2.
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f2: Non-reducing (without β-mercaptoethanol) SDS–PAGE analysis of UV-C irradiated HGDC samples (1 mg/ml) at different exposure times (M: protein marker, Lane 1: 0 min, Lane 2: 10 min, Lane 3: 20 min, Lane 4: 30 min, Lane 5: 40 min, Lane 6: 50 min, Lane 7: 60 min). The dose of UV-C irradiation used was 1,850 μW/cm2.

Mentions: To identify the size distribution of species obtained by exposure of HGDC to UV-C irradiation, the irradiated (at 1,850 μW/cm2) HGDC samples at 1.0 mg/ml, taken at different times (0, 10, 20, 30, 40, 50, and 60 min), were electrophoresed on SDS–PAGE under non-reducing conditions (without β-mercaptoethanol). We demonstrate in Figure 2 that a prominent band at ~21 kDa, belonging to the monomeric species, and a slightly vague (a faint) band, corresponding to the dimeric species (see Figure 2, lane 1), were detected for the HGDC sample at t=0. When subjected to UV-C irradiation, the HGDC samples showed evidence of aggregated species, probably the dimer and oligomer, that migrated with molecular weights greater than ~42 kDa on the gel (e.g.: 20 min, see Figure 2, lane 3). Upon further irradiation, the bands corresponding to the aforesaid aggregated species became more apparent and a certain amount of macromolecular species was visibly trapped in the wells of the stacking gel (e.g.: 20 ~60 min, see Figure 2, lane 3–7). Moreover, as shown in Figure 2, lanes 2–3, some of the HGDC monomers were converted into HGDC dimeric species at the early stage of UV-C irradiation (e.g., ~20 min). However, as revealed by lanes 4–7 of Figure 2, further exposure to UV-C irradiation led to a significant reduction in the amount of HGDC dimers, which is most likely attributed to the involvement of dimeric species in the oligomerization or polymerization process.


Examining the influence of ultraviolet C irradiation on recombinant human γD-crystallin.

Wang SS, Wen WS - Mol. Vis. (2010)

Non-reducing (without β-mercaptoethanol) SDS–PAGE analysis of UV-C irradiated HGDC samples (1 mg/ml) at different exposure times (M: protein marker, Lane 1: 0 min, Lane 2: 10 min, Lane 3: 20 min, Lane 4: 30 min, Lane 5: 40 min, Lane 6: 50 min, Lane 7: 60 min). The dose of UV-C irradiation used was 1,850 μW/cm2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008712&req=5

f2: Non-reducing (without β-mercaptoethanol) SDS–PAGE analysis of UV-C irradiated HGDC samples (1 mg/ml) at different exposure times (M: protein marker, Lane 1: 0 min, Lane 2: 10 min, Lane 3: 20 min, Lane 4: 30 min, Lane 5: 40 min, Lane 6: 50 min, Lane 7: 60 min). The dose of UV-C irradiation used was 1,850 μW/cm2.
Mentions: To identify the size distribution of species obtained by exposure of HGDC to UV-C irradiation, the irradiated (at 1,850 μW/cm2) HGDC samples at 1.0 mg/ml, taken at different times (0, 10, 20, 30, 40, 50, and 60 min), were electrophoresed on SDS–PAGE under non-reducing conditions (without β-mercaptoethanol). We demonstrate in Figure 2 that a prominent band at ~21 kDa, belonging to the monomeric species, and a slightly vague (a faint) band, corresponding to the dimeric species (see Figure 2, lane 1), were detected for the HGDC sample at t=0. When subjected to UV-C irradiation, the HGDC samples showed evidence of aggregated species, probably the dimer and oligomer, that migrated with molecular weights greater than ~42 kDa on the gel (e.g.: 20 min, see Figure 2, lane 3). Upon further irradiation, the bands corresponding to the aforesaid aggregated species became more apparent and a certain amount of macromolecular species was visibly trapped in the wells of the stacking gel (e.g.: 20 ~60 min, see Figure 2, lane 3–7). Moreover, as shown in Figure 2, lanes 2–3, some of the HGDC monomers were converted into HGDC dimeric species at the early stage of UV-C irradiation (e.g., ~20 min). However, as revealed by lanes 4–7 of Figure 2, further exposure to UV-C irradiation led to a significant reduction in the amount of HGDC dimers, which is most likely attributed to the involvement of dimeric species in the oligomerization or polymerization process.

Bottom Line: The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride.Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC.The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan. sswang@ntu.edu.tw

ABSTRACT

Purpose: Human γD crystallin is a principal protein component of the human eye lens and associated with the development of juvenile and mature-onset cataracts. Exposure to ultraviolet (UV) light is thought to perturb protein structure and eventually lead to aggregation. This work is aimed at exploring the effects of UV-C irradiation on recombinant human γD-crystallin (HGDC).

Methods: Recombinant HGDC proteins were expressed in E. coli strain BL21(DE3) harboring plasmid pEHisHGDC and purified using chromatographic methods. The proteins were then exposed to UV-C light (λ(max)=254 nm, 15 W) at the intensity of 420, 800, or 1850 μW/cm(2). The UV-C-unexposed, supernatant fraction of UV-C-exposed, and re-dissolved precipitated fraction of UV-C exposed preparations were characterized by SDS-PAGE, turbidity measurement, CD spectroscopy, tryptophan fluorescence spectroscopy, acrylamide fluorescence quenching analysis, and sulfhydryl group measurements.

Results: The turbidity of the HGDC sample solution was found to be positively correlated with HGDC concentration, UV-C irradiation intensity, and UV-C irradiation duration. When exposed to UV-C, HGDC sample solutions became visibly turbid and a noticeable amount of larger protein particle, perceptible to the naked eye, was observed upon prolonged irradiation. The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride. Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC. Through the use of L-cysteine, the measurements of sulfhydryl contents, and the reducing as well as non-reducing SDS-PAGE, our data further suggested that disulfide bond formation and/or cleavage probably play an important role in aggregation and/or precipitation of HGDC elicited by UV-C irradiation.

Conclusions: Our findings highlight the close connections among disulfide bond cleavage and/or formation, intermolecular interactions, and the resultant formation of aggregates of HGDC induced by UV-C irradiation. The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.

Show MeSH
Related in: MedlinePlus