Diabetes mellitus (DM) is a chronic metabolic disorder characterized by inappropriate hyperglycemia, which causes endothelial dysfunction and peripheral neuropathy, ultimately leading to multiple complications. One prevalent complication is diabetic erectile dysfunction (ED), which is more severe and more resistant to treatment than nondiabetic ED. The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1) is a modulator of TGF-β-mediated angiogenesis and has been proposed as a biomarker for a variety of diseases, including DM. Here, we found that the adhesion GPCR latrophilin-2 (LPHN2) is a TGF-β-independent receptor of LRG1. By interacting with LPHN2, LRG1 promotes both angiogenic and neurotrophic processes in mouse tissue explants under hyperglycemic conditions. Preclinical studies in a diabetic ED mouse model showed that LRG1 administration into the penile tissue, which exhibits significantly increased LPHN2 expression, fully restores erectile function by rescuing vascular and neurological abnormalities. Further investigations revealed that PI3K, AKT, and NF-κB p65 constitute the key intracellular signaling pathway of the LRG1/LPHN2 axis, providing important mechanistic insights into LRG1-mediated angiogenesis and nerve regeneration in DM. Our findings suggest that LRG1 can be a potential new therapeutic option for treating aberrant peripheral blood vessels and neuropathy associated with diabetic complications, such as diabetic ED.
Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein−protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni 2+ and Cu 2+ ), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni 2+ . In addition, when Cu 2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.
The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1), primarily produced by hepatocytes and neutrophils, is a multifunctional protein that modulates various signaling cascades, mainly TGFβ signaling. Serum LRG1 and neutrophil-derived LRG1 have different molecular weights due to differences in glycosylation, but the impact of the differential glycan composition in LRG1 on its cellular function is largely unknown. We previously reported that LRG1 can promote both angiogenic and neurotrophic processes under hyperglycemic conditions by interacting with LPHN2. Here, we determined the crystal structure of LRG1, identifying the horseshoe-like solenoid structure of LRG1 and its four N-glycosylation sites. In addition, our biochemical and cell-biological analyses found that the deglycosylation of LRG1, particularly the removal of glycans on N325, is critical for the high-affinity binding of LRG1 to LPHN2 and thus promotes LRG1/LPHN2-mediated angiogenic and neurotrophic processes in mouse tissue explants, even under normal glucose conditions. Moreover, the intracavernous administration of deglycosylated LRG1 in a diabetic mouse model ameliorated vascular and neurological abnormalities and restored erectile function. Collectively, these data indicate a novel role of LRG1 glycans as molecular switches that can tune the range of LRG1’s cellular functions, particularly the LRG1/LPHN2 signaling axis.
The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1), primarily produced by hepatocytes and neutrophils, is a multifunctional protein that can modulate various signalling cascades, mainly TGFβ signalling. Serum LRG1 and neutrophil-derived LRG1 have different molecular weights due to differences in glycosylation, but what impact the differential glycan composition in LRG1 has on its cellular function is largely unknown. We previously reported that LRG1 can promote both angiogenic and neurotrophic processes under hyperglycemic conditions by interacting with LPHN2. Here, we determined the crystal structure of LRG1, identifying the horseshoe-like solenoid structure of LRG1 and its four N-glycosylation sites. In addition, our biochemical and cell-biological analysis found that de-glycosylation of LRG1, particularly the removal of glycans on N325, is critical for high-affinity binding of LRG1 to LPHN2, thereby promoting LRG1/LPHN2-mediated angiogenic and neurotrophic processes in mouse tissue explants, even under normal glucose conditions. Moreover, intracavernous administration of de-glycosylated LRG1 in a diabetic mouse model ameliorated vascular and neurological abnormalities and restored erectile function. Collectively, these data indicate a novel role of LRG1’s glycans as molecular switches that can tune the range of LRG1’s cellular functions, particularly the LRG1/LPHN2 signalling axis.
: Based on the activated sludge model(ASM), a mathematical model which represents the aerobic sludge digestion by sequencing batch reactor(SBR) combined with ultrasonic treatment was composed and performed in this study. Aerobic digestion using sequencing batch reactor(SBR) equipped with ultrasound treatment was also experimented for the purpose of parameter calibration. Most of the presented kinetic parameters in ASM or ASM2 could be used for the aerobic digestion of sludge but the parameters related in hydrolysis and decay rate needed modification. Hydrolysis rate constant of organic matter in aerobic condition was estimated at 0.3 day -1 and the maximum growth rate for autotrophs in aerobic condition was 0.618 day -1 . Solubilization reactions of particulate organics and nitrogen by ultrasonication was added in this kinetic model. The solubilization rate is considered to be proportional to the specific energy which is defined by specific ultrasound power and sonication time. The solubilization rate constant by ultrasonication was estimated at 0.202(W/L) -1 day -1 in this study. Autotrophs as well as heterotrophs also decomposed by ultrasonic treatment and the nitrification reaction was limited by the lack of autotrophs accumulation in the digester.
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