[Posterior arthroscopic subtalar arthrodesis for symptomatic adult talocalcaneal coalition].

Objective: To investigate the functional outcomes of posterior arthroscopic subtalar arthrodesis (PASTA) for adult patients presenting with symptomatic talocalcaneal coalition. Methods: The study was a retrospective case-series research.The data of 17 adult patients (17 feet) with symptomatic talocalcaneal coalitions,treated with PASTA from March 2018 to February 2022 in Xuzhou Central Hospital were collected.This procedure involved 10 males and 7 females,aged (42.4±7.5) years(range:31 to 58 years).There were 9 cases on the right side and 7 cases on the left side.According to the Rozansky classification,there were 4 cases of type Ⅰ,7 cases of type Ⅱ, 3 cases of type Ⅲ,3 cases of type Ⅳ.The following items such as wound healing and bony union of the subtalar joint were observed.Clinical assessment was performed using pain visual analogue scale (VAS),American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot scores and 36-item short form health survey (SF-36) scores.The paired t test was used for data comparison. Results: The follow-up time was (24.8±6.9) months(range:12 to 40 months).There were no complications such as wound infection,deep vein thrombosis,nonunion,or screw breakage.One patient with preoperative spasm,relieved after the second surgical procedure (peroneal brevis tendon lengthening).The union time of the subtalar joint was (8.8±2.2) weeks(range:6 to 12 weeks).At the final follow-up,the VAS decreased from (6.4±1.3) to (1.3±0.9)(t=14.114,P<0.01), the AOFAS ankle-hindfoot score increased from (49.0±8.1) to (90.0±5.1)(t=38.782,P<0.01),and the SF-36 score increased from (50.8±9.5) to (91.0±4.9)(t=20.468,P<0.01). Conclusion: PASTA for adult patients presenting with symptomatic talocalcaneal coalition offers advantages of minimal trauma,fast recovery,and few complications,which is an effective method.

[Arthroscopic treatment of acute closed noninsertional rupture of Achilles tendon].

Objective: To investigate the clinical efficacy of arthroscopic treatment of acute closed noninsertional rupture of Achilles tendon. Methods: The clinical and imaging data of 30 patients (30 feet) with acute closed noninsertional rupture of Achilles tendon who were treated with all-inside arthroscopic technique at the Department of Hand and Foot Microsurgery,Xuzhou Central Hospital from June 2018 to June 2020 were analyzed retrospectively. There were 26 males and 4 females,aged (38.3±8.5)years old(range:19 to 66 years). There were 22 cases on the right side and 8 cases on the left side. The duration from injury to surgery was (2.1±1.4) days (range:1 to 7 days).All patients were treated with all-inside arthroscopic technique.The function of the ankle and the foot was assessed using visual analogue scale (VAS),the American Orthopaedic Foot & Ankle Society (AOFAS) ankle hindfoot scale and the Achilles tendon total rupture score (ATRS). The Arner-Lindholm score system was used to evaluate the excellent and good rate of clinical effect. Paired sample t test or rank-sum test was used for data comparison. Results: The patients were followed up for (18.6±2.2)months(range:12 to 28 months).All the wounds healed at the first stage.No complication such as infection,sural nerve injury or re-rupture happened.Two patinets felt mild pain after a long time exercise, and were alleviated by microwave therapy and stretching the Achilles tendon consistently.Another patient was unable to do a sustained single stance heel raise,which was recovered after repeated function practice.At the last follow-up,the VAS (M(IQR)) decreased from 6(5) preoperatively to 0(1)(Z=6.512,P<0.01),the AOFAS ankle hindfoot scale improved from 60.6±8.3 preoperatively to 96.3±4.8(t=-29.774,P<0.01),and the ATRS improved from 61.7±7.8 preoperatively to 97.1±2.3 (t=-53.661,P<0.01).According to the Arner-Lindholm score system,27 cases were excellent,3 cases were good,and the excellent and good rate was 100%. Conclusions: The all-inside arthroscopic technique not only ensures the quality of tendon ananstomosis,but also avoids injury to the sural nerve.It has the advantages of small trauma,faster recovery and fewer complications.

Rapid personalized chemotherapy recommendation for cancer patients: An interview with Shaohua Ma and two members of his group.

Shaohua Ma, an early-career group leader, and his team talk about their passion for data science and their project published in Patterns, where multiplex gene quantification-based "digital markers" are used for extremely rapid evaluation of chemo-drug sensitivity. This method allows quick and personalized chemo-drug recommendations for cancer patients, helping to improve their clinical care and health outcomes.

[The reconstruction of Myerson type â ¢ chronic Achilles tendon rupture by using the total arthroscopic technique combined with free semitendinosus tendon and gracilis tendon autograft].

Objective: To evaluate the clinical outcome of the reconstruction of Myerson type Ⅲ chronic Achilles tendon rupture by using the total arthroscopic technique combined with free semitendinosus tendon and gracilis tendon autograft. Methods: Clinical data of 32 patients(32 ankles) with Myerson type Ⅲ chronic Achilles tendon rupture who were treated by using the total arthroscopic technique combined with free semitendinosus tendon and gracilis tendon autograft at Department of Hand and Foot Microsurgery, Xuzhou Central Hospital from September 2013 to September 2018 were analyzed retrospectively.There were 28 males and 4 females, aged 45.5 years old(range: 22 to 69 years old), 12 cases in the right side and 20 in the left.All patients were treated by using the total arthroscopic technique combined with free semitendinosus tendon and gracilis tendon autograft for Myerson type Ⅲ chronic Achilles tendon rupture reconstruction.The functional recovery of the ankle was evaluated according to ankle-hindfood score of American Orthopaedic Foot and Ankle Society (AOFAS) Ankle Hindfoot Scale, Achilles tendon total rupture score (ATRS), visual analogue scale (VAS).Arner-Lindholm score was used to evaluate the excellent and good rate.The quantitative data were compared using t-test or Wilcoxon test. Results: The 32 patients were followed up for 33 months (range: 15 to 72 months).No serious postoperative complications such as infection, sural nerve injury or tend re-rupture outcomes were reported.Three patients complained of mild pain when after a minimum sitting, walking or jogging, which were completely relieved by simple physical therapy or continuous stretching of Achilles tendon.At the last follow-up, the VAS decreased from 3 (5) (M (Q(R)) ) preoperative to 0 (3) (Z=1.357, P<0.01) and AOFAS ankle hindfoot scale improved from 58.6±13.5 preoperative to 95.5±4.0 (t=16.9, P=0.00), ATRS improved from 47.5±9.3 preoperative to 96.6±3.3 (t=25.661, P<0.01).According to the score of Arner-Lindholm, 20 cases were excellent, 12 cases were good, and the excellent and good rate was 100%. Conclusion: The reconstruction of Myerson type Ⅲ chronic Achilles tendon rupture by using the total arthroscopic technique combined with free semitendinosus tendon and gracilis tendon autograft has the advantages of safety, reliability, effectiveness and minor injury.

[Arthroscopy-assisted percutaneous reduction and screw fixation for displaced intra-articular calcaneal fractures].

Objective: To investigate the surgical technology and clinical efficacy of using the all-inside arthroscopic treatment of intra-articular displaced calcaneal fracture. Methods: A retrospective analysis was made of 46 patients (46 feet) with intra-articular displaced calcaneal fractures treated by modified all-inside arthroscopic from December 2015 to December 2017 in Xuzhou Central Hospital. Twenty-eight cases (28 feet) were male and 18 cases (18 feet) were female, aged from 19 to 60 years (mean (39±13) years). The time from injury to operation was 1 to 10 days (mean (4.5±2.2) days). The pre-operative visual analogue scale (VAS) of pain ranged from 4 to 8 points, mean (6.3±1.8) points. No other combined injuries were found in the affected feet in all patients. All patients were treated with anterolateral, lateral and posterolateral approaches of arthroscopically assisted reduction for calcaneal fractures, and fixed with hollow screw. Main outcome measures included the pain, foot appearance, and the scores of the American Orthopedic Foot & Ankle Society (AOFAS) Scale and the Maryland Scale. Paired t test was used when the data before and after the operation were compared. Results: All incisions healed in one stage without postoperative complications such as nerve, vessel and tendon injury. The mean follow-up period ranged from 12 to 36 months, mean (21±9) months. Postoperatively X-ray showed complete fracture healing time was 8 to 12 weeks, mean (10.4±2.3) weeks. At the last follow-up, the ankle and talocalcaneal joints movement and appearance were ideal, and no traumatic arthritis was found. The VAS score of talocalcaneal joint was 0. AOFAS score increased from 58±13 to 96±9 and Maryland score increased from 72±11 to 98±8 after the operation, the differences were all significant (t=15.958, 12.496, both P<0.05). Conclusion: The all-inside arthroscopic treatment of the intra-articular displaced calcaneal fracture is an effective and precise method, with accurately outcomes, precise reduction and minimally postoperative complications.

[Analysis of the effect of two medial portals for the all-inside endoscopic treatment of recalcitrant plantar fasciitis].

Objective: To examine the clinical effect of all-inside endoscopic treatment of recalcitrant plantar fasciitis through two medial portals. Methods: The recalcitrant plantar fasciitis data of 67 cases (79 feet) that underwent two medial portals all-inside endoscopic treatment at Department of Hand and Foot Microsurgery, Xuzhou Central Hospital from October 2016 to June 2018 were retrospectively analyzed.There were 24 males (30 feet) and 43 females (49 feet) aged 44.3 years old(range:24-76 years).The mean disease duration from the specialist doctor intervention to operation was (23.7±11.0) months (range: 12-60 months). All the patients were treated with the two medial portals all-inside endoscopic procedure when the 6 months conservative treatment had failed.The endoscopic procedure including debridement and partial plantar fasciotomy.The clinical results,including pain,activity,gait and foot health quality,were scored using visual analogue pain scale (VAS),American Orthopaedic Foot & Ankle Society Ankle Hindfoot Scale (AOFAS) and SF-36. Results: All the patients were followed up for (15.2±6.7) months (range: 12-24 months). All cases achieved primarily healing of the wound without postoperative complications of nerve,vessel and tendon.At the last follow-up,the VAS decreased from (5.3±2.0) preoperative to 0 prooperative (t=21.60, P=0.000), AOFAS increased from (72.6±9.4) to (97.3±4.6)(t=19.43,P=0.000),SF-36 increased from (93.6±8.4) to (119.1±7.3) (t=18.78, P=0.000), non-recurrent calcaneal spur, normal foot and ankle activity was recorded. Conclusion: The two medial portals all-inside endoscopic procedure is effective for the treatment of recalcitrant plantar fasciitis.

Clinical effect of E-series of prostaglandin receptor 2 and epidermal growth factor receptor signal pathways in the development of esophageal squamous cell carcinoma.

Signal pathways mediated by epidermal growth factor receptor (EGFR) and E-series of prostaglandin receptors (EPs) are closely correlated to the pathogenesis of tumor. This experiment was designed to investigate the expression and clinical significance of EP2 and EGFR in esophageal squamous cell carcinoma (ESCC). Tissue samples were collected reterospectively from 87 patients with ESCC (first diagnosed). The patients were followed up for 5 years after radical surgery. The expression of EP-2 and EGFR were examined by tissue chip technology and immunohistochemistry methods. Clinicopathological and prognostic impact were evaluated. Overexpression of EGFR and EP-2 was more observed in ESCC than the control group (58.6% vs. 13.9%; 52.9% vs. 4.88%, P < 0.001, respectively); which correlated with tumor infiltration depth, lymph node metastasis, and tumor-lymph node-metastasis staging. Both the EP-2 and EGFR overexpression were detected in 39 specimens and exhibited the positive correlation (P < 0.001, r = 0.404). Overexpression of EP2 and EGFR exhibited significant correlation with worse 5-year overall survival than those with negative result (17.6% vs. 27.8%, P = 0.011; 10.9% vs. 34.1%, P < 0.001, respectively). Cox proportional hazard model showed that the T-staging, lymph node metastasis, and EGFR overexpression were the independent risk factors of the prognosis. The present study exhibited that the overexpression of EP2 and EGFR in ESCC tissues might play an important role in carcinogenesis and the progression of ESCC.

Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation.

The establishment of high-efficiency Agrobacterium-mediated transformation techniques could improve the production of Dioscorea zingiberensis, a medicinal species with a high diosgenin content. We co-cultivated embryogenic calli induced from mature seeds with A. tumefaciens strain EHA105. A binary vector, pCAMBIA1381, which contains the gfp and hpt genes under the control of the ubiquitin promoter and the CaMV 35S promoter, respectively, was used for transformation. Pre-culture, basic medium, acetosyringone, and bacterial density were evaluated to establish the most efficient protocol. The optimal conditions consisted of MS medium without CaCl(2) for pre- and co-cultivation, three days for pre-culture, addition of 200 µM AS, and an OD(600) of 0.5. The transgenic plants grown under selection were confirmed by PCR analysis and Southern blot analysis. This protocol produced transgenic D. zingiberensis plants in seven months, with a transformation efficiency of 6%.

Pharmacological chaperone therapy by active-site-specific chaperones in Fabry disease: in vitro and preclinical studies.

Many genetic disorders are due to protein misfolding and excessive premature degradation in the endoplasmic reticulum (ER). When a gene mutation does not affect the functionality of the protein, it may still promote the premature clearance of the protein by ER-associated degradation (ERAD), resulting in a loss of function. Competitive inhibitors are often effective active-site-specific chaperones when used at sub-inhibitory concentrations. Active-site-specific chaperones assist in the folding of mutant lysosomal enzymes in the ER, thereby promoting their escape from ERAD, enhancing trafficking to the lysosome and increasing the level of residual enzyme activity. In Fabry disease, degradation of various mutant forms of a-galactosidase A (alpha-gal A) has been shown to take place in the ER as a result of protein misfolding. One of the most potent inhibitors of alpha-gal A, 1-deoxygalactonojirimycin, has also been shown to be effective in enhancing residual alpha-gal A activity in cultured fibroblasts and lymphoblasts established from patients with Fabry disease caused by a variety of missense mutations. Oral administration of 1-deoxygalactonojirimycin to transgenic mice expressing a mutant form of human alpha-gal A (R301Q) yielded higher alpha-gal A activity in major tissues, compared with untreated transgenic mice.

Suppression of PPN/MG61 attenuates Wnt/beta-catenin signaling pathway and induces apoptosis in human lung cancer.

Wingless and int homologue (Wnt) family proteins have been shown to have important roles in the decision of cell fate and behavior at multiple stages during the development and tumorigenesis. One of the Drosophila segment polarity genes, porcupine (porc) gene, encodes an evolutionarily conserved endoplasmic reticulum membrane protein involving in the post-translational processing of the Wnt family proteins. Here, we report that human homologue of Drosophila porc gene, PPN/MG61, was abundantly expressed in human cancer cell lines, but not in normal cells. We also found that PPN/MG61 was overexpressed in primary lung cancer tissue samples, compared to their matched normal tissue samples. Furthermore, when we used small interfering RNA to knock down PPN/MG61 mRNA in lung cancer cells expressing the gene, we observed apoptosis induction, along with decreased activity of Wnt pathway in those lung cancer cells. These data suggest that PPN/MG61 may be a novel marker for human lung cancer and that post-translational modification of the Wnt signal molecules by PPN/MG61 may be important for the function of Wnt pathway in lung cancer.

Novel alpha-L-fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae).

The extract of bark of Angylocalyx pynaertii (Leguminosae) was found to potently inhibit mammalian alpha-L-fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 1-deoxymannojirimycin (DMJ) and 2,5-imino-1,2,5-trideoxy-D-mannitol (6-deoxy-DMDP). Among them, eight sugar-mimic alkaloids showed the potent inhibitory activity towards bovine epididymis alpha-L-fucosidase and their Ki values are as follows: 6-deoxy-DMDP (83 microM), 2,5-imino-1,2,5-trideoxy-L-glucitol (0.49 microM), 2,5-dideoxy-2,5-imino-D-fucitol (17 microM), 2,5-imino-1,2,5-trideoxy-D-altritol (3.7 microM), DMJ (4.7 microM), N-methyl-DMJ (30 microM), 6-O-alpha-L-rhamnopyranosyl-DMJ (Rha-DMJ, 0.06 microM), and beta-L-homofuconojirimycin (beta-HFJ, 0.0053 microM). We definitively deduced the structural requirements of inhibitors of alpha-L-fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of alpha-L-fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of L-fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of L-fucose generates extremely powerful inhibition of alpha-L-fucosidase. The replacement of the methyl group of beta-HFJ by a hydroxymethyl group reduced its inhibitory potential about 80-fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of L-fucose does not appear to be important for the inhibition. Interestingly, Rha-DMJ was a 70-fold more potent inhibitor of alpha-L-fucosidase than DMJ. This implies that the lysosomal alpha-L-fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates.

In vitro inhibition and intracellular enhancement of lysosomal alpha-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives.

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal alpha-galactosidase A (alpha-Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal alpha-galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1-deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.-Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med. 5, 112-115]. 1-Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1-deoxygalactonojirimycin derivatives were tested for activity with both alpha-Gal A and Fabry lymphoblasts. 1-Deoxygalactonojirimycin was the most potent inhibitor of alpha-Gal A with an IC50 value of 0.04 microM. alpha-Galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 microM, respectively. N-Alkylation, deoxygenation at C-2 and epimerization at C-3 of 1-deoxygalactonojirimycin markedly lowered or abolished its inhibition toward alpha-Gal A. Inclusion of 1-deoxygalactonojirimycin, alpha-galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin at 100 microM in culture medium of Fabry lymphoblasts increased the intracellular alpha-Gal A activity by 14-fold, 5.2-fold, 2.4-fold and 2.3-fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of alpha-Gal A are effective specific chemical chaperones for Fabry disease.

Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease.

Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.

Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor.

Fabry disease is a disorder of glycosphingolipid metabolism caused by deficiency of lysosomal alpha-galactosidase A (alpha-Gal A), resulting in renal failure along with premature myocardial infarction and strokes. No effective treatment of this disorder is available at present. Studies of residual activities of mutant enzymes in many Fabry patients showed that some of them had kinetic properties similar to those for normal alpha-Gal A, but were significantly less stable, especially in conditions of neutral pH (refs. 3-5). The biosynthetic processing was delayed in cultured fibroblasts of a Fabry patient, and the mutant protein formed an aggregate in endoplasmic reticulum, indicating that the enzyme deficiency in some mutants was mainly caused by abortive exit from the endoplasmic reticulum, leading to excessive degradation of the enzyme. We report here that 1-deoxy-galactonojirimycin (DGJ), a potent competitive inhibitor of alpha-Gal A, effectively enhanced alpha-Gal A activity in Fabry lymphoblasts, when administrated at concentrations lower than that usually required for intracellular inhibition of the enzyme. DGJ seemed to accelerate transport and maturation of the mutant enzyme. Oral administration of DGJ to transgenic mice overexpressing a mutant alpha-Gal A substantially elevated the enzyme activity in some organs. We propose a new molecular therapeutic strategy for genetic metabolic diseases of administering competitive inhibitors as 'chemical chaperons' at sub-inhibitory intracellular concentrations.

Enzymatic synthesis of a neoglycoconjugate by transglycosylation with Arthrobacter endo-beta-N-acetylglucosaminidase: a substrate for colorimetric detection of endo-beta-N-acetylglucosaminidase activity.

The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenyl-alpha-D-glucose (Man6GlcNAc-Glc-pNP). The reaction was efficiently induced in aqueous solution containing dimethyl sulfoxide. In the medium containing 20% (v/v) dimethyl sulfoxide with 0.1 M Glc-pNP as an acceptor, the transglycosylation attained yields of 75% by high-performance anion-exchange chromatography. The structure of Man6GlcNAc-Glc-pNP was confirmed by ion mass spectrometry and 400 MHz 1H NMR spectrometry. Various endo-beta-N-acetylglucosaminidases hydrolyzed this oligosaccharide and Man6GlcNAc and Glc-pNP were released from the oligosaccharide by endo-beta-N-acetylglucosaminidase digestion. We have established a new procedure for the colorimetric detection of endo-beta-N-acetylglucosaminidase activity using Man6Glc-NAc-Glc-pNP, which is simple as that for other exoglycosidase assays with pNP-glycosides as substrates.

Detailed studies on substrate structure requirements of glycoamidases A and F.

Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively. A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.

Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells.

Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein (BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG. Following purification of the immunoglobulins using Protein A-Sepharose, glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoamidase from sweet almond emulsin to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with 2-aminopyridine and the structures identified by two-dimensional high performance liquid chromatography mapping (Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, 73-90). The N-glycans obtained from the secreted IgG contain 35% complex type, some with terminal galactose residues at either alpha1, 3-Man or alpha1,6-Man branches of the Man3GlcNAc2 core. The remaining oligosaccharides detected in the secreted IgG were principally hybrid (30%) and paucimannosidic (35%) type N-glycans. Most (84%) of these secreted glycoforms contained fucose alpha1, 6-linked to the innermost GlcNAc residue and the presence of a potentially allergenic fucose alpha1,3-linked to the innermost GlcNAc residue was also detected. In contrast, the intracellular immunoglobulins included 50% high mannose-type N-glycans with lower levels of complex, hybrid, and paucimannosidic-type structures. Reverse phase one-dimensional high performance liquid chromatography analysis of the IgG N-glycans in the absence of heterologous BiP exhibited a similar distribution of intracellular and secreted glycoforms. These studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processing pathways in these cell lines appear to diverge from mammalian cells in the formation of paucimannosidic structures, in the presence of alpha1,3-fucose linkages, and in the absence of sialylation.

Transfer of Man9GlcNAc to L-fucose by endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

We have reported that transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man9GlcNAc, to L-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation produce was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man9GlcNAc-L-Fuc-PA, revealed that a beta-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man9GlcNAc-L-Fuc; and ii) preparation of Man9GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man9GlcNAc beta (1,2)-L-Fuc. Methyl-beta-L-fucopyranoside, L-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl alpha-L-fucopyranoside, D-Fuc and D-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.

Purification of 2-aminopyridine derivatives of oligosaccharides and related compounds by cation-exchange chromatography.

A simple method for efficient removal of a large excess of 2-aminopyridine from the reaction mixture of pyridylamination of carbohydrates and related compounds by cation-exchange chromatography is presented. The method is especially suitable for purification and separation of small-sized pyridylaminated-(PA-) derivatives and large-scale preparations. Separation of PA-derivatives from 2-aminopyridine was accomplished by a Dowex 50X8 column (NH+4-form) eluted with 20 mM ammonium acetate buffer (pH 8.5). The PA derivatives thus purified can be used for high-performance liquid chromatography analysis directly. The method requires only 10 min to purify a sample, in contrast to an hour or longer required by the gel filtration method. Unlike the existing methods for purification method. Unlike the existing methods for purification of PA-oligosaccharides, which are applicable only in the range of 0.05-100 nmol, the cation-exchange method allows processing of much larger quantities of PA-derivatives. For example, PA-L-glyceraldehyde (10 mumol) was successful purified by a Dowex 50X8 column (1.5 x 17 cm) eluted with 20 mM ammonium acetate buffer (pH 8.75) within 3 h.