Endo-beta-N-acetylglucosaminidase: Possible Functions and Mechanisms / 生物化学与生物物理进展

Endo-beta-N-acetylglucosaminidase (ENGase) is widely distributed in various organisms. The first reported ENGase activity was detected in Diplococcus pneumoniae in 1971. The protein (Endo D) was purified and its peptide sequence was determined in 1974. Three ENGases (Endo F1-F3) were discovered in Flavobacterium meningosepticum from 1982 to 1993. After that, the activity was detected from different species of bacteria, yeast, fungal, plant, mice, human, etc. Multiple ENGases were detected in some species, such as Arabidopsis thaliana and Trichoderma atroviride. The first preliminary crystallographic analysis of ENGase was conducted in 1994. But to date, only a few ENGases structures have been obtained, and the structure of human ENGase is still missing. The currently identified ENGases were distributed in the GH18 or GH85 families in Carbohydrate-Active enZyme (CAZy) database. GH18 ENGase only has hydrolytic activity, but GH85 ENGase has both hydrolytic and transglycosylation activity. Although ENGases of the two families have similar (β/α)8-TIM barrel structures, the active sites are slightly different. ENGase is an effective tool for glycan detection andglycan editing. Biochemically, ENGase can specifically hydrolyze β‑1,4 glycosidic bond between the twoN-acetylglucosamines (GlcNAc) on core pentasaccharide presented on glycopeptides and/or glycoproteins. Different ENGases may have different substrate specificity. The hydrolysis products are oligosaccharide chains and a GlcNAc or glycopeptides or glycoproteins with a GlcNAc. Conditionally, it can use the two products to produce a new glycopeptides or glycoprotein. Although ENGase is a common presentation in cell, its biological function remains unclear. Accumulated evidences demonstrated that ENGase is a none essential gene for living and a key regulator for differentiation. No ENGase gene was detected in the genomes of Saccharomyces cerevisiae and three other yeast species. Its expression was extremely low in lung. As glycoproteins are not produced by prokaryotic cells, a role for nutrition and/or microbial-host interaction was predicted for bacterium produced enzymes. In the embryonic lethality phenotype of the Ngly1-deficient mice can be partially rescued by Engase knockout, suggesting down regulation of Engase might be a solution for stress induced adaptation. Potential impacts of ENGase regulation on health and disease were presented. Rabeprazole, a drug used for stomach pain as a proton inhibitor, was identified as an inhibitor for ENGase. ENGases have been applied in vitro to produce antibodies with a designated glycan. The two step reactions were achieved by a pair of ENGase dominated for hydrolysis of substrate glycoprotein and synthesis of new glycoprotein with a free glycan of designed structure, respectively. In addition, ENGase was also been used in cell surface glycan editing. New application scenarios and new detection methods for glycobiological engineering are quickly opened up by the two functions of ENGase, especially in antibody remodeling and antibody drug conjugates. The discovery, distribution, structure property, enzymatic characteristics and recent researches in topical model organisms of ENGase were reviewed in this paper. Possible biological functions and mechanisms of ENGase, including differentiation, digestion of glycoproteins for nutrition and stress responding were hypothesised. In addition, the role of ENGase in glycan editing and synthetic biology was discussed. We hope this paper may provide insights for ENGase research and lay a solid foundation for applied and translational glycomics.

Allergy Associated With N-glycans on Glycoprotein Allergens / 生物化学与生物物理进展

Protein as the allergens could lead to allergy. In addition, a widespread class of allergens were known as glycans of N-glycoprotein. N-glycoprotein contained oligosaccharide linked by covalent bonds with protein. Recently,studies implicated that allergy was associated with glycans of heterologous N-glycoprotein found in food, inhalants, insect toxins, etc. The N-glycan structure of N-glycoprotein allergen has exerted an influence on the binding between allergens and IgE, while the recognition and presentation of allergens by antigen-presenting cells (APCs) were also affected. Some researches showed thatN-glycan structure of allergen was remodeled by N-glycosidase, such as cFase I, gpcXylase, as binding of allergen and IgE partly decreased. Thus, allergic problems caused by N-glycoproteins could potentially be solved by modifying or altering the structure ofN-glycoprotein allergens, addressing the root of the issue. Mechanism of N-glycans associated allergy could also be elaborated through glycosylation enzymes, alterations of host glycosylation. This article hopes to provide a separate insight for glycoimmunology perspective, and an alternative strategy for clinical prevention or therapy of allergic diseases.

Lower limb deep vein ultrasonography and blood biochemistry in women after full-term delivery / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the postpartum changes in lower limb deep vein ultrasonography and blood biochemistry in women 2-5 days after full-term delivery.</p><p><b>METHODS</b>A total of 212 women at high risk of thrombosis underwent high-resolution color Doppler ultrasound (CDU) of the lower limb deep veins 2-5 days after full-term delivery (Group A). Sixty-one healthy women 2-5 days after full-term delivery (Group B) and 42 healthy non-pregnant women (Group C) were recruited as the controls. The hematological indexes including the D-dimers (D-D), platelet (PLT), hemoglobin (HGB), hematocrit (HCT), thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (Fbg) were also determined in these 3 groups.</p><p><b>RESULTS</b>Compared to Group B, the women in group A showed significantly increased diameters and D-D value of the common femoral veins (FV), common superficial femoral veins (SFV) and common popliteal veins (POV) (P<0.01), with decreased peak blood flow in the bilateral POVs (P<0.05). Compared to those in Groups C, the diameters of the bilateral FVs, SFVs, POVs, and posterior tibial veins (PTVs) were significantly increased, but the peak blood flow in the bilateral FVs, SFVs, and POVs were significantly reduced in groups A and B; the PLT, HGB, HCT, DD, TT, APTT, PT, and Fbg also showed significant changes in groups A and B (P<0.01).</p><p><b>CONCLUSIONS</b>The women 2-5 days after full-term delivery are at high risk of prethrombotic state in comparison with non-pregnant women, and the presence of high-risk factors for thrombosis increases the likeliness of prothrombotic state in these postpartum women. CDU examination of the lower limb deep veins can be of value in the diagnosis of prethrombotic state.</p>

Inhibitory effects of RNA interference on MMP-24 expression and invasiveness of ovarian cancer SKOV(3) cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of RNA interference (RNAi) on MMP-24 expression and invasiveness of ovarian cancer SKOV(3) cells.</p><p><b>METHOD</b>Two pairs of small interfering RNA (siRNA) specific to MMP-24 mRNA were designed and transfected into SKOV(3) cells. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of MMP-24, and the cell invasiveness was assessed using an in vitro invasion test.</p><p><b>RESULTS</b>After transfection with siRNA, the mRNA and protein expression levels of MMP-24 were obviously reduced in SKOV(3) cells, which also showed significantly decreased invasiveness in vitro.</p><p><b>CONCLUSIONS</b>MMP-24 gene silencing by RNAi can suppress the invasiveness of ovarian cancer SKOV(3) cells in vitro, which may provide a new therapeutic approach of ovarian cancer.</p>

Expression of ZNF217 in human ovarian cystadenocarcinoma and its clinical significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the correlation of ZNF217 expression to the carcinogenesis and progression of human ovarian cancer.</p><p><b>METHODS</b>Immunohistochemistry and real-time RT-PCR were used to detect ZNF217 expression in human ovarian cystadenocarcinoma, ovarian cystadenoma and normal ovary tissues.</p><p><b>RESULTS</b>The expression levels of ZNF217 protein and mRNA in ovarian cystadenocarcinoma was significantly higher than those in matched ovarian cystadenoma and normal tissues (P<0.05). No significant difference was found in the expression between ovarian cystadenoma and normal ovarian tissues (P>0.05). The mRNA expression in the specimens was consistent with the protein expression of ZNF217 (P<0.05).</p><p><b>CONCLUSION</b>ZNF217 gene expression is closely correlated to the occurrence and clinical stages of ovarian carcinomas, suggesting that ZNF217 can be an important candidate gene responsible for the occurrence and progression of ovarian carcinomas.</p>

Ultrasonic study of deep-vein diameter and blood flow spectrum changes in full-term pregnant women / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the changes in lower limb deep vein diameters, blood flow velocity and blood biochemistry in full-term pregnant women for early diagnosis and treatment of prothrombotic state.</p><p><b>METHODS</b>One hundred and twenty-eight full-term pregnant women at high risk of thrombosis (Group A), 61 healthy full-term pregnant women (Group B), and 42 healthy non-pregnant women (Group C) underwent high-resolution color Doppler ultrasound (CDU) for examining the deep veins of the lower limbs. The hematological indexes such as D-D, PLT, HGB, HCT, TT, APTT, PT, and FbgC were also observed in these 3 groups.</p><p><b>RESULTS</b>Compared to Group B, the women in group A showed significantly increased diameters of the common femoral veins (CFV) and left superficial femoral vein (SFV), HCT and DD, but with significantly decreased peak blood flow in the bilateral popliteal veins (POPV) (P<0.01) and increased left POPV diameter (P=0.034). Compared to those in group C, the diameters of the bilateral CFVs, SFVs, POPV, and posterior tibial veins (PTVs) were significantly increased, but the peak blood flow in the bilateral CFVs and POPVs were significantly reduced in groups A and B; the PLT, HGB, HCT, DD, TT, APTT, PT, and FbgC also showed significant changes in groups A and B (P<0.01).</p><p><b>CONCLUSION</b>The full-term pregnant women are at higher risk of prothrombotic state than non-pregnant women, and the full-term pregnant women with the high risk factors for thrombosis are more likely to have prothrombotic state than healthy full-term pregnant women. CDU examination of the lower limb deep veins can be of value in the diagnosis of prothrombotic state.</p>

Establishment of a nude mouse model bearing orthotopically transplanted human ovarian carcinoma expressing green fluorescent protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a human ovarian cancer-bearing mouse model via orthotopic transplantation of human HO-8910 cells expressing green fluorescent protein (GFP).</p><p><b>METHODS</b>GFP-expressing human ovarian carcinoma HO8910/GFP cells (2 x 10(6)) in exponential phase of growth were inoculated subcutaneously in nude mice, and the generated tumor tissues were collected and transplanted below the capsule of the left ovary of 6 nude mice. The growth of the tumors was observed in vivo using a fluorescence stereomicroscope. The nude mice were sacrificed 4 weeks after transplantation to assess the tumor growth and metastasis.</p><p><b>RESULTS</b>The tumors showed progressive growth at the orthotopic sites in all animals. Two weeks after the transplantation, green fluorescent mass was observed at the left costovertebral angle, and the mass increased thereafter and invaded or metastasized to the peritoneum, omentum, spleen, liver, uterus, and the pelvic lymph nodes, with a metastatic rate as much as 66.7%.</p><p><b>CONCLUSION</b>The nude mouse model bearing orthotopic human ovarian carcinoma expressing GFP has been successfully established.</p>

A clinicopathologic study of dysembryoplstic neuroepithelial tumor / 中华病理学杂志

<p><b>UNLABELLED</b>OBJECTIVE To study the clinicopathologic features, radiologic findings, treatment modalities and prognosis of dysembryoplastic neuroepithelial tumor (DNT).</p><p><b>METHODS</b>The clinical features, histopathologic findings, immunohistochemistry and electron microscopy of 18 cases of DNT were analyzed. Results Among the 18 cases studied, 14 were males and 4 females. The age of these patients ranged from 3 to 46 (mean age = 22. 8 years). Partial seizure was the main presenting symptom in all patients. The history of epilepsy could be as long as 17 years. On magnetic resonance imaging (MRI) study, the tumor was hypodense on T1 and hyperdense on T2. There was neither edema nor mass effect. All but 2 cases were supratentorial and intracortical in location. Ten cases were treated by complete surgical excision and the remaining 8 tumors were partially excised. In the 14 patients with follow-up data available, 13 survived for 1.4 to 11 years after the operation (with more than 10 years survival observed in 2 patients). The average survival period was 5.5 years. None of the cases showed tumor recurrence after operation. Histologically, all tumors demonstrated a multinodular architecture and were intracortical in location, sometimes with extension into the white matter. The characteristic "glioneuronal constituent" was an essential feature for making the diagnosis of DNT. The tumor was formed by an admixture of oligodendrocyte-like cells, mature neurons and astrocytes, with obvious microcystic changes. These neurons were often dispersed singly in the mucoid matrix. In most cases, the foci of cortical dysplasia were found in adjacent areas. Immunohistochemical study demonstrated positivity for synaptophysin, neurofilament and S-100 protein in the neurons and some oligodendrocyte-like cells. The staining of glial fibrillary acidic protein in the oligodendrocyte-like cells was negative. Electron microscopy showed early neuronal, astrocytic and oligodendroglial differentiation of the oligodendrocyte-like cells.</p><p><b>CONCLUSIONS</b>DNT is a benign tumor (corresponding to WHO grade I) that can be cured by surgical excision, despite sometimes incomplete tumor removal. A correct diagnosis of this entity requires thorough understanding of the clinical, radiologic, histologic and immunohistochemical features.</p>

Study on the properties of a novel glycine amino peptidase from Actinomucor elegans / 生物工程学报

The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE-Toyopearl column, a Toyopearl HW 65-C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14.2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56.5kD by SDS-PAGE. It hydrolyzes glycine residue containing substrates such as glycine-betanaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly-betaNA, the enzyme could also hydrolyze Ala-betaNA, Met-betaNA, Leu-betaNA, Arg-betaNA and Ser-betaNA but it had no activity on the substrates such as Trp-betaNA, Pyr-betaNA, Pro-betaNA, Asp-betaNA, Lys-betaNA, Val-betaNA. It was also observed when the glycine-betanaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine-betanaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30 degrees C and pH 8.0, respectively. The Km and Kcat of the enzyme for glycine-betanaphthylamine is 0.24 mmol/L and 100.8 s(-1), respectively. Zn2+, Cu2+ and Cd2+ suppress almost all activities of the enzyme at the concentration of 1.0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0.5% of alkaline proteinase together with glycine aminopeptidase at 50 degrees C for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual amino acids analysis with an amino acid analyzer showed that the contents of glycine, proline, alanine, arginine and glutamate were considerably increased. The results of this study showed that the glycine aminopeptidase would be useful in the food industry.