To date, few structural models of VHH antibody binding to low molecular weight haptens have been reported. Here, we report the crystal structure of cortisol binding to its VHH antibody NbCor at pH 3.5 and 10.5. Cortisol binds to NbCor mainly by burying itself under the tunnel formed by the complementarity determining region 1 (CDR1) of NbCor. The affinity of NbCor binding to cortisol and similar compounds was also verified by a microscale thermophoresis assay. Combining our findings with several previously reported structures of hapten‐VHH antibody complexes, we propose that VHH antibodies exhibit a special mechanism of binding small haptens by encapsulating them in a tunnel formed by CDR1. Our findings provide useful structural information for the further development and optimization of hapten‐specific VHH antibodies.
Accumulation of α-synuclein (α-Syn) is a remarkable pathology for Parkinson’s disease (PD), therefore clearing it is possibly a promising strategy for treating PD. Aberrant copper (Cu(II)) homeostasis and oxidative stress play critical roles in the abnormal aggregation of α-Syn in the progress of PD. It is reported that the polyphenol (−)-epi-gallocatechin gallate (EGCG) can inhibit α-Syn fibrillation and aggregation, disaggregate α-Syn mature fibrils, as well as protect α-Syn overexpressed-PC12 cells against damage. Also, previous studies have reported that EGCG can chelate many divalent metal ions. What we investigate here is whether EGCG can interfere with the Cu(II) induced fibrillation of α-Syn and protect the cell viability. In this work, on a molecular and cellulaire basis, we demonstrated that EGCG can form a Cu(II)/EGCG complex, leading to the inhibition of Cu(II)-induced conformation transition of α-Syn from random coil to β-sheet, which is a dominant structure in α-Syn fibrils and aggregates. Moreover, we found that the mixture of Cu(II) and EGCG in a molar ratio from 0.5 to 2 can efficiently inhibit this process. Furthermore, we demonstrated that in the α-Syn transduced-PC12 cells, EGCG can inhibit the overexpression and fibrillation of α-Syn in the cells, and reduce Cu(II)-induced reactive oxygen species (ROS), protecting the cells against Cu(II)-mediated toxicity.
Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future.
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