Proteome and Glycoproteome Analyses Reveal Regulation of Protein Glycosylation Site-Specific Occupancy and Lysosomal Hydrolase Maturation by N-Glycan-Dependent ER-Quality Control.

N-Glycan-dependent endoplasmic reticulum quality control (ERQC) primarily mediates protein folding, which determines the fate of the polypeptide. Monoglucose residues on N-glycans determine whether the nascent N-glycosylated proteins enter into and escape from the calnexin (CANX)/calreticulin (CALR) cycle, which is a central system of the ERQC. To reveal the impact of ERQC on glycosylation and protein fate, we performed comprehensive quantitative proteomic and glycoproteomic analyses using cells defective in N-glycan-dependent ERQC. Deficiency of MOGS encoding the ER α-glucosidase I, CANX, or/and CALR broadly affected protein expression and glycosylation. Among the altered glycoproteins, the occupancy of oligomannosidic N-glycans was significantly affected. Besides the expected ER stress, proteins and glycoproteins involved in pathways for lysosome and viral infection are differentially changed in those deficient cells. We demonstrated that lysosomal hydrolases were not correctly modified with mannose-6-phosphates on the N-glycans and were directly secreted to the culture medium in N-glycan-dependent ERQC mutant cells. Overall, the CANX/CALR cycle promotes the correct folding of glycosylated peptides and influences the transport of lysosomal hydrolases.

Sialylation on vesicular integrin ß1 determined endocytic entry of small extracellular vesicles into recipient cells.

BACKGROUND: Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. METHODS: Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin ß1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. RESULTS: (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin ß1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin ß1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin ß1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. CONCLUSIONS: Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1, which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1. LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells.

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modified methods for LC isolation and compared their performance with that of the regular method. Modified method 1 integrated the crude LCs into the 50% Percoll solution before centrifugation. Modified method 2 sequentially used 50 and 60% Percoll solutions to isolate LCs. The purity of LCs was approximately 88.4, 91.3, and 79.7% derived from the regular, modified 1, and modified 2 methods, respectively. The yields of LCs in the same respective order were approximately 1.7 × 105, 3.9 × 105, and 11.9 × 105 cells per 108 interstitial cells input. Modified method 1 attained higher purity and yields than those of the regular method. Although the purity of LCs was relatively low for modified method 2, it could be used before further purification by, for example, fluorescence-activated or magnetic-activated cell sorting, owing to its simplicity and high yields. Therefore, our study provided alternative methods to facilitate LC isolation in mice.

Bioinspired, Ultra-Fast Polymerization of Dopamine under Mild Conditions.

Spontaneous oxidative polymerization of dopamine (DA) is widely exploited as a facile and versatile method for surface modification. However, the reaction is very slow and only occurs in alkaline solutions, which severely limit its applications. Herein it is reported that the reaction can be dramatically accelerated by using Fe2+ as catalyst. While it takes hours and days using conventional method, the Fe2+ -catalyzed reaction finishes almost immediately at pH 7.0. In addition, under the catalysis of Fe2+ , the reaction can occur at a pH down to 4.0. The fast Fe2+ -catalyzed polymerization of DA leads to fast deposition of polydopamine (PDA) coating, thus allowing fast surface modification and textile dyeing. The Fe2+ -catalyzed reaction also allows spatial control over the PDA deposition. The fast, simple, and mild surface modification method developed here will find applications in numerous fields.

An Activatable Near-Infrared Fluorescence Hydrogen Sulfide (H2S) Donor for Imaging H2S Release and Inhibiting Inflammation in Cells.

Hydrogen sulfide (H2S) is a vital endogenous signal molecule that exerts critical physiological functions such as biological regulation and cytoprotection. Despite significant progress in developing H2S donors, site-specific delivery and controllable release of H2S in biological systems remain a key challenge. Herein, we develop new Cys-triggered fluorescent H2S donor Pro-S that is composed of a dicyanoisophorone-based near-infrared (NIR) fluorescent dye and a thiocarbamate moiety. The H2S donor releases H2S under the attack of Cys, accompanied by the release of a fluorescent reporter, which enables the real-time capturing of H2S by fluorescence spectroscopy or microscopy. Pro-S exhibits strong NIR fluorescence enhancement (70-fold), excellent controllable H2S release (30 min), high H2S release efficiency (62%), and well live-cell compatibility, allowing for visualization of H2S release in cells and zebrafish. Moreover, Pro-S presents a good effect of anti-inflammation in RAW 264.7 cells. This work provides a new idea for the design of H2S donors, which may be beneficial to the comprehension of the potential mechanism of inflammation and optimization of treatment strategies.

LINC01414/LINC00824 genetic polymorphisms in association with the susceptibility of chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a complicated multi-factor, multi-gene disease. Here, we aimed to assess the association of genetic polymorphisms in LINC01414/ LINC00824 and interactions with COPD susceptibility. METHODS: Three single nucleotide polymorphisms (SNPs) in LINC01414/LINC00824 was genotyped by Agena MassARRAY platform among 315 COPD patients and 314 controls. Logistic analysis adjusted by age and gender were applied to estimate the genetic contribution of selected SNPs to COPD susceptibility. RESULTS: LINC01414 rs699467 (OR = 0.73, 95% CI 0.56-0.94, p = 0.015) and LINC00824 rs7815944 (OR = 0.56, 95% CI 0.31-0.99, p = 0.046) might be protective factors for COPD occurrence, while LINC01414 rs298207 (OR = 2.88, 95% CI 1.31-6.31, p = 0.008) risk-allele was related to the increased risk of COPD in the whole population. Rs7815944 was associated with the reduced risk of COPD in the subjects aged > 70 years (OR = 0.29, p = 0.005). Rs6994670 (OR = 0.57, p = 0.007) contribute to a reduced COPD risk, while rs298207 (OR = 7.94, p = 0.009) was related to a higher susceptibility to COPD at age ≤ 70 years. Rs298207 (OR = 2.54, p = 0.043) and rs7815944 (OR = 0.43, p = 0.028) variants was associated COPD risk among males. Rs7815944 (OR = 0.16, p = 0.031) was related to the reduced susceptibility of COPD in former smokers. Moreover, the association between rs298207 genotype and COPD patients with dyspnea was found (OR = 0.50, p = 0.016), and rs7815944 was related to COPD patients with wheezing (OR = 0.22, p = 0.008). CONCLUSION: Our finding provided further insights into LINC01414/LINC00824 polymorphisms at risk of COPD occurrence and accumulated evidence for the genetic susceptibility of COPD.

Sialidase NEU1 suppresses progression of human bladder cancer cells by inhibiting fibronectin-integrin α5ß1 interaction and Akt signaling pathway.

BACKGROUND: Sialic acids are widely distributed in animal tissues, and aberrantly expressed in a variety of cancer types. High expression of sialic acid contributes to tumor aggressiveness by promoting cell proliferation, migration, angiogenesis, and metastasis. Sialidases are responsible for removal of sialic acids from glycoproteins and glycolipids. METHODS: N-glycomics of bladder cancer cells were detected by MALDI-TOF mass spectrometry. Sialic acid modification in bladder cancer tissue was determined by lectin blot. The down-regulation of NEU1 in bladder cancer cells was determined by high resolution liquid chromatography mass spectrometry (HR LC-MS). The effects of sialidase NEU1 expression on proliferation and apoptosis of human bladder cancer cells were examined by western blot, RT-PCR, confocal imaging and flow cytometry. Moreover, the function of sialic acids on fibronectin-integrin α5ß1 interaction were assayed by immunoprecipitation and ELISA. The importance of NEU1 in tumor formation in vivo was performed using BALB/c-nu mice. Expression of NEU1 in primary human bladder cancer tissue samples was estimated using bladder cancer tissue microarray. RESULTS: (1) Downregulation of NEU1 was primarily responsible for aberrant expression of sialic acids in bladder cancer cells. (2) Decreased NEU1 expression was correlated with bladder cancer progression. (3) NEU1 overexpression enhanced apoptosis and reduced proliferation of bladder cancer cells. (4) NEU1 disrupted FN-integrin α5ß1 interaction and deactivated the Akt signaling pathway. (5) NEU1 significantly suppressed in vivo tumor formation in BALB/c-nu mice. CONCLUSIONS: Our data showed that NEU1 inhibited cancer cell proliferation, induced apoptosis, and suppressed tumor formation both in vitro and in vivo, by disrupting interaction of FN and integrin ß1 and inhibiting the Akt signaling pathway. Our observations indicate that NEU1 is an important modulator of the malignant properties of bladder cancer cells, and is a potential therapeutic target for prognosis and treatment of bladder cancer. Video Abstract.

EPIDEMIOLOGIC CHARACTERISTICS AND RISK FACTORS FOR CONGENITAL HYPOTHYROIDISM FROM 2009 TO 2018 IN XIAMEN, CHINA.

Objective: Early diagnosis and treatment of children with congenital hypothyroidism (CH) through newborn screening can effectively prevent delayed development. This study was designed to investigate the pathogenesis and factors that influence CH in urban areas of China between 2009 and 2018. Methods: A retrospective analysis of newborn screening data and diagnosis and treatment information for CH diagnosed in the information database of the neonatal disease screening center in one of China's five special economic zones from 2009 to 2018. Results: Of the 947,258 newborns screened between 2009 and 2018, 829 (406 girls) were diagnosed with CH at birth (1 diagnosis/1,136 births). Among the 608 cases of CH diagnosed at birth and re-evaluated at the age of 3 years, 487 were permanent congenital hypothyroidism (PCH, 1/1,429), and 121 were transient congenital hypothyroidism (TCH, 1/5,882). A total of 83.2% of infants with PCH (405/487) underwent thyroid imaging in the neonatal period, of which thyroid dysgenesis accounted for 28.64% (116/405) and functional defects accounted for 71.36% (289/405). The incidence of CH changed significantly in infants with initial serum thyroid-stimulating hormone concentrations of 41 to 100 mIU/L and ≥100 mIU/L, whereas the incidence of mild CH showed a slight increase. The incidence of CH was significantly higher in postterm infants (1/63) and low-birth-weight infants (1/370). Conclusion: In the past decade, the incidence of CH has increased, mainly due to the increase in the incidence of PCH and TCH. The incidence of mild CH has increased slightly. Postterm birth and low birth weight are important factors affecting the incidence of CH. Abbreviations: CH = congenital hypothyroidism; FT4 = free thyroxine; L-T4 = levothyroxine sodium; PCH = permanent congenital hypothyroidism; TCH = transient congenital hypothyroidism; TSH = thyroid-stimulating hormone; TT4 = total thyroxine.

Telomere length, ZNF208 genetic variants and risk of chronic obstructive pulmonary disease in the Hainan Li population.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a disease characterized by airflow limitation. It is not completely reversible and shows progressive development. ZNF208 rs8105767 affects telomere length, although the impact of telomere on COPD is still controversial. In the present study, we aimed to explore the impact of the ZNF208 gene polymorphism on telomere length and also that of telomere length on COPD in the Hainan Li population. METHODS: In total, 270 COPD patients and 288 controls were recruited. Telomere length was measured by a quantitative real-time polymerase chain reaction. Five single nucleotide polymorphisms in ZNF208 were selected and genotyping was performed using MassARRAY software (Agena Bioscience Co. Ltd, San Diego, CA, USA). Differences in telomere length among the subjects with three genotypes of related genes were assessed using analysis of variance. Unconditional logistic regression was used to calculate odds ratios (OR) as the indicator of association between telomere length and COPD risk. RESULTS: Relative telomere length in the COPD group and control group was 0.66 ± 0.47 and 1.44 ± 0.89, respectively. We grouped according to a median of 0.8284 for telomere length and observed that the risk of COPD for individuals with a telomere length less than 0.8284 is 2.92 times that for individuals with a telomere length longer than 0.8284 (OR = 2.92, 95% confidence interval = 2.01-4.25, p = 1.91 × 10-8 ). Subjects carrying the G allele of rs2188972 had a longer telomere length. Subjects carrying the carrying the CA genotype of rs8103163 and AC genotype of rs7248488 had a longer telomere length compared to wild-type individuals. CONCLUSIONS: Shorter telomeres increase COPD risk and the ZNF208 polymorphism affects telomere length in COPD patients.

Conditioned Medium from Malignant Breast Cancer Cells Induces an EMT-Like Phenotype and an Altered N-Glycan Profile in Normal Epithelial MCF10A Cells.

Epithelial-mesenchymal transition (EMT) is a key process in cancer development and progression. Communication (crosstalk) between cancer cells and normal (nonmalignant) cells may facilitate cancer progression. Conditioned medium (CM) obtained from cultured cancer cells contains secreted factors capable of affecting phenotypes and the behaviors of normal cells. In this study, a culture of normal breast epithelial MCF10A cells with CM from malignant breast cancer cells (termed 231-CM and 453-CM) resulted in an alteration of morphology. CM-treated MCF10A, in comparison with control cells, showed a reduced expression of the epithelial marker E-cadherin, increased expression of the mesenchymal markers fibronectin, vimentin, N-cadherin, and TWIST1, meanwhile cell proliferation and migration were enhanced while cell apoptosis was decreased. N-glycan profiles of 231-CM-treated and control MCF10A cells were compared by MALDI-TOF/TOF-MS (Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry) and a lectin microarray analysis. The treated cells showed lower levels of high-mannose-type N-glycan structures, and higher levels of complex-type and hybrid-type structures. Altered N-glycan profiles were also detected in 453-CM-treated and non-treated MCF10A cells by MALDI-TOF/TOF-MS, and we found that the expression of five fucosylated N-glycan structures (m/z 1406.663, 1590.471, 1668.782, 2421.141, and 2988.342) and one high-mannose structure m/z 1743.722 have the same pattern as 231-CM-treated MCF10A cells. Our findings, taken together, show that CM derived from breast cancer cells induced an EMT-like process in normal epithelial cells and altered their N-glycan profile.

Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions.

Lipoxygenase (LOX; EC 1.13.11.12) is an enzyme which is widely used in food industry to improve aroma and rheological or baking properties of foods. A series of studies have proven that the flexible regions negatively relates to the thermal stability of enzymes. In this study, two highly flexible regions, residues(20-49) and residues(201-206), were modified to improve the thermal stability of LOX from Pseudomonas aeruginosa. Deletion of the first 20 and 30 residues of the former region increased the thermal stability of the LOX by 1.3- and 2.1-fold, respectively. Although deletion of the residues(201-206) led to a sharp reduction of both thermal stability and catalytic activity of the enzyme, the residue substitutions with the glycines (G204P, G206P, and G204P/G206P) or even glycine-rich linker (L6/PT) within this region increased the thermal stability of LOX by values ranging from 0.46- to 3.45-fold. To be noted, over 85% of the specific activity was maintained in all thermally stabilized LOX mutants. Circular dichroism and fluorescence analysis showed that the overall secondary and tertiary structures were not significantly changed by these modifications. To the best of our knowledge, this is the first report on increasing the thermal stability of LOX by protein engineering without remarkably affecting the catalytic rate.

Enhancement of thermostability and expression level of Rasamsonia emersonii lipase in Pichia pastoris and its application in biodiesel production in a continuous flow reactor.

The acidic lipase from Rasamsonia emersonii named LIPR has great potential for biodiesel synthesis due to its strong methanol tolerance. Nonetheless, the limited thermostability of LIPR and low expression level in Escherichia coli remain major obstacles to its use in biodiesel synthesis. To enhance the thermostability, the mutant LIPR harboring mutations A126C-P238C for the formation of a new disulfide bond and amino acid substitution D214L was obtained through rational design. To our delight, the thermostability of LIPR mutant was greatly improved. Moreover, a comprehensive optimization strategy, such as employing the Mss signal peptide, co-expressing the molecular chaperone protein disulfide isomerase (PDI), knocking out the vacuolar sorting receptor gene VPS10-01, and overexpressing the dihydroxyacetone synthase gene DAS2, was adopted to obtain the combination-optimized mutant Pichia pastoris strain GS54. Furthermore, the biodiesel synthetic capability with the mutant GS54-LIPR was verified and the production yield was 52.2 % after 24 h in a shake flask. Subsequently, a continuous flow system was adopted to increase the biodiesel yield to 73.6 % within 3 h, demonstrating its efficacy in enhancing enzyme biocatalysis. The engineered GS54-LIPR mutant lipase is an efficient and reusable biocatalyst for the sustained production of biodiesel in a continuous flow reaction.

Protein Signatures for Distinguishing Colorectal Cancer Liver Metastases from Primary Liver Cancer Using Tissue Slide Proteomics.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.

A case report of using gauze packing to treat postoperative chest bleeding after left pneumonectomy for secondary rifampicin-resistant tuberculosis.

One patient with rifampin-resistant tuberculosis underwent emergency left pneumonectomy and thoracic gauze packing for hemoptysis due to recurrent hemoptysis after transcatheter arterial embolization. Vital signs were maintained by mechanical ventilation and medication. Tracheotomy and anti-tuberculosis treatment were performed. After half a year of follow-up, the patient's condition was stable.

Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides.

Self-assembling amphipathic peptides (SAPs) are a category of peptides that have unique sequences with alternating hydrophobic and hydrophilic residues that can spontaneously assemble into ordered nanostructures. In this study, we investigated the potential of fusion technique with SAPs to improve the thermal stability of lipoxygenase (LOX) from Pseudomonas aeruginosa. Six SAPs were individually fused to the N terminus of the LOX that resulted to the SAP-LOX fusions with approximately 2.3- to 4.5-fold enhanced thermal stability at 50 °C. The specific activities of the SAP-LOX fusions were also increased by 1.0- to 2.8-fold as compared with the wild-type LOX. This is the first report on the improvement of the thermal stability and specific activity of an enzyme by the fused SAPs, suggesting a simple technique to improve the catalytic properties of the recombinant enzymes by fusion expression.

Sialylation: A Cloak for Tumors to Trick the Immune System in the Microenvironment.

The tumor microenvironment (TME), where the tumor cells incite the surrounding normal cells to create an immune suppressive environment, reduces the effectiveness of immune responses during cancer development. Sialylation, a type of glycosylation that occurs on cell surface proteins, lipids, and glycoRNAs, is known to accumulate in tumors and acts as a "cloak" to help tumor cells evade immunological surveillance. In the last few years, the role of sialylation in tumor proliferation and metastasis has become increasingly evident. With the advent of single-cell and spatial sequencing technologies, more research is being conducted to understand the effects of sialylation on immunity regulation. This review provides updated insights into recent research on the function of sialylation in tumor biology and summarizes the latest developments in sialylation-targeted tumor therapeutics, including antibody-mediated and metabolic-based sialylation inhibition, as well as interference with sialic acid-Siglec interaction.

Expression of a Siglec-Fc Protein and Its Characterization.

The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism.

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar L-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of D-fructose, L-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce L-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, L-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by L-sorbose-treatment. Thus, L-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, L-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate L-sorbose as an attractive therapeutic reagent for cancer treatment.

Site- and Structure-Specific Glycosylation Signatures of Bovine, Caprine, Porcine, and Human Milk-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.