Glycation
between proteins and reducing sugars is the common chemical
modification in food protein, and many studies have focused on the
allergenicity of the glycated protein. However, a systemic study on
the allergenicity change of its digests is lacking. In this work,
we explored the change rule of the digestibility and allergenicity
of glycated β-Lg during in
vitro gastrointestinal digestion and interpreted the mechanism using high-resolution
mass spectrometry. Glycation with arabinose increased the resistance
of β-Lg to digestive enzyme, with a low hydrolysis value. Indirect
competitive ELISA showed that the IgG/IgE binding rates of β-Lg
were reduced after glycation and further reduced after digestion,
in comparison with the digests of unglycated β-Lg. There are
two reasons for this phenomenon. On the one hand, 11 glycated sites
were determined in the lowest allergenicity arabinose-β-Lg conjugation
(Ara-β-Lg), which was distributed in the IgG and IgE linear
allergic epitopes of β-Lg. On the other hand, glycation masking
linear allergenic epitopes had a more significant effect on reducing
allergenicity in comparison to digestive enzyme hydrolysis. These
results indicated that the allergenicity of Ara-β-Lg in the
human body might be lower than that of unglycated β-Lg.
Trefoil (3(1)) and figure-8 (4(1)) knots have been synthesized from DNA molecules containing two single-turn helical domains, linked by four oligodeoxythymidine linkers. Both topologies are derived from the same DNA molecule. The tightest knots are fashioned by minimizing the lengths of the linkers. The shortest equal-length linkers from which a trefoil knot can be made readily are seven nucleotides long, in a 74-nucleotide molecule, whereas those in the shortest figure-8 knot are six nucleotides long, in a 70-nucleotide molecule. In addition to these limiting knots, other knots containing 80, 88, 96 and 104 nucleotides have been constructed. The mobilities of these molecules on denaturing gels show the conventional logarithmic dependence on length. Ferguson analysis of their mobilities indicates a linear dependence of surface area on length. The 80-mer trefoil knot is the tightest molecule that can be restricted in both domains.
Our previous study indicated that pretreatment by dynamic high-pressure microfluidization (DHPM) and glycation with galactose was a promising method for decreasing the immunoglobulin E (IgE)-binding ability of β-lactoglobulin (β-LG). In this work, the conformational alteration of β-LG subjected to DHPM and glycation treatment was investigated in relation to IgE-binding ability by orbitrap mass spectrometry. After DHPM pretreatment, lower IgE-binding ability of glycated β-LG was observed with increasing pressures. Prior to DHPM pretreatment, 11 glycated sites were identified, while the number of glycation sites was increased to 12 after pretreatment. However, there was no significant difference of the glycation sites at the pressures of 50, 100, and 200 MPa, respectively. Average degree of substitution per peptide molecule of β-LG (DSP) was investigated to assess the degree of glycation per glycation site. All of the samples pretreated by DHPM exhibited a higher glycation level than those without DHPM pretreatment. The shielding effects of epitopes owing to glycation contributed to the reduction of IgE-binding capacity. Orbitrap mass spectrometry could provide a comprehensive understanding of the nature of protein glycation.
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