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Variations in LOXL1 associated with exfoliation glaucoma do not affect amine oxidase activity.

Kim S, Kim Y - Mol. Vis. (2012)

Bottom Line: However, there were no significant differences in amine oxidase activity between the LOXL1 haplotype variants toward the tested substrates.The R141L and G153D variations in the NH(2)-terminal region of LOXL1 do not affect the amine oxidase activity of LOXL1.Our results suggest that other unknown genetic factors or molecular mechanisms may be more relevant to the development of XFG.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, Wonkwang University, Sinyong-Dong 344-2, Iksan-City, Jeollabuk-Do 570-749, South Korea.

ABSTRACT

Purpose: Lysyl oxidase-like 1 (LOXL1) is a copper-dependant amine oxidase that plays an essential role in elastogenesis. Two non-synonymous single-nucleotide polymorphisms of LOXL1, R141L (rs1048661) and G153D (rs3825942), have been reported to significantly increase susceptibility to exfoliation glaucoma (XFG). To evaluate the impact of the R141L and G153D variations on the amine oxidase activity of LOXL1, we generated four different haplotypes of LOXL1 with R141L and G153D and assessed the amine oxidase activity of the LOXL1 variant proteins.

Methods: The four different haplotype variants of LOXL1 were created by oligonucleotide-directed mutagenesis in an LOXL1 expression vector. Recombinant LOXL1 variant proteins were purified by nickel-affinity chromatography. The amine oxidase activities of the LOXL1 variant proteins were assessed using peroxidase-coupled fluorometric assays.

Results: All of the haplotype variants of LOXL1 (141R-153G, 141R-153D, 141L-153G, and 141L-153D) showed β-aminopropionitrile-inhibitable amine oxidase activity toward elastin, type I collagen, and cadaverine, indicating that each LOXL1 variant functions as an amine oxidase. However, there were no significant differences in amine oxidase activity between the LOXL1 haplotype variants toward the tested substrates.

Conclusions: The R141L and G153D variations in the NH(2)-terminal region of LOXL1 do not affect the amine oxidase activity of LOXL1. This is consistent with recent genetic findings on the reversal of risk alleles of R141L and G153D in different ethnic backgrounds. Our results suggest that other unknown genetic factors or molecular mechanisms may be more relevant to the development of XFG.

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Expression and purification of the four different haplotype proteins of LOXL1. Each lane contains approximately 3 μg of purified recombinant LOXL1 protein of the 141L-153G, 141L-153D, 141R-153G, or 141R-153D haplotype. Lane M contains a molecular mass standard.
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f2: Expression and purification of the four different haplotype proteins of LOXL1. Each lane contains approximately 3 μg of purified recombinant LOXL1 protein of the 141L-153G, 141L-153D, 141R-153G, or 141R-153D haplotype. Lane M contains a molecular mass standard.

Mentions: Upon induction with 1 mM IPTG in E. coli, all expression constructs of the four different LOXL1 variants showed high levels of expression. However, the LOXL1 variant proteins were within inclusion bodies. The insoluble fractions were solubilized with 6 M urea, and then the LOXL1 variant proteins were purified by nickel-chelating affinity chromatography using a hexa-histidine tag attached at the COOH-termini of the variant proteins. To refold the LOXL1 variant proteins denatured by urea during purification, the protein samples were subjected to stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The apparent sizes of the purified recombinant LOXL1 variant proteins were in good agreement with the deduced molecular mass, 56 kDa, and were over 95% pure on SDS–PAGE gels (Figure 2).


Variations in LOXL1 associated with exfoliation glaucoma do not affect amine oxidase activity.

Kim S, Kim Y - Mol. Vis. (2012)

Expression and purification of the four different haplotype proteins of LOXL1. Each lane contains approximately 3 μg of purified recombinant LOXL1 protein of the 141L-153G, 141L-153D, 141R-153G, or 141R-153D haplotype. Lane M contains a molecular mass standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275637&req=5

f2: Expression and purification of the four different haplotype proteins of LOXL1. Each lane contains approximately 3 μg of purified recombinant LOXL1 protein of the 141L-153G, 141L-153D, 141R-153G, or 141R-153D haplotype. Lane M contains a molecular mass standard.
Mentions: Upon induction with 1 mM IPTG in E. coli, all expression constructs of the four different LOXL1 variants showed high levels of expression. However, the LOXL1 variant proteins were within inclusion bodies. The insoluble fractions were solubilized with 6 M urea, and then the LOXL1 variant proteins were purified by nickel-chelating affinity chromatography using a hexa-histidine tag attached at the COOH-termini of the variant proteins. To refold the LOXL1 variant proteins denatured by urea during purification, the protein samples were subjected to stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The apparent sizes of the purified recombinant LOXL1 variant proteins were in good agreement with the deduced molecular mass, 56 kDa, and were over 95% pure on SDS–PAGE gels (Figure 2).

Bottom Line: However, there were no significant differences in amine oxidase activity between the LOXL1 haplotype variants toward the tested substrates.The R141L and G153D variations in the NH(2)-terminal region of LOXL1 do not affect the amine oxidase activity of LOXL1.Our results suggest that other unknown genetic factors or molecular mechanisms may be more relevant to the development of XFG.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, Wonkwang University, Sinyong-Dong 344-2, Iksan-City, Jeollabuk-Do 570-749, South Korea.

ABSTRACT

Purpose: Lysyl oxidase-like 1 (LOXL1) is a copper-dependant amine oxidase that plays an essential role in elastogenesis. Two non-synonymous single-nucleotide polymorphisms of LOXL1, R141L (rs1048661) and G153D (rs3825942), have been reported to significantly increase susceptibility to exfoliation glaucoma (XFG). To evaluate the impact of the R141L and G153D variations on the amine oxidase activity of LOXL1, we generated four different haplotypes of LOXL1 with R141L and G153D and assessed the amine oxidase activity of the LOXL1 variant proteins.

Methods: The four different haplotype variants of LOXL1 were created by oligonucleotide-directed mutagenesis in an LOXL1 expression vector. Recombinant LOXL1 variant proteins were purified by nickel-affinity chromatography. The amine oxidase activities of the LOXL1 variant proteins were assessed using peroxidase-coupled fluorometric assays.

Results: All of the haplotype variants of LOXL1 (141R-153G, 141R-153D, 141L-153G, and 141L-153D) showed β-aminopropionitrile-inhibitable amine oxidase activity toward elastin, type I collagen, and cadaverine, indicating that each LOXL1 variant functions as an amine oxidase. However, there were no significant differences in amine oxidase activity between the LOXL1 haplotype variants toward the tested substrates.

Conclusions: The R141L and G153D variations in the NH(2)-terminal region of LOXL1 do not affect the amine oxidase activity of LOXL1. This is consistent with recent genetic findings on the reversal of risk alleles of R141L and G153D in different ethnic backgrounds. Our results suggest that other unknown genetic factors or molecular mechanisms may be more relevant to the development of XFG.

Show MeSH
Related in: MedlinePlus