Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability.
-glycans are important for mucin ...stability and function, but their structures have not been fully characterized, unlike that of
-glycans. In this study, BSM
-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32
-glycans were identified, and the quantities (%) of each
-glycan relative to total
-glycans (100%) were obtained. The terminal
-acetylgalactosamines in 12
-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated
-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present
-glycans and reported
-glycans). No
-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM
-glycans. Mucin structures, including sulfated
-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated
-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.
Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (
-glycans) are important in regulating BSM's viscoelasticity and polymerization. However, details of
...-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of
-glycans and identifies
-glycosylation sites in BSM using liquid chromatography-tandem mass spectrometry. The
-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total
-glycans (100%; 1.1 pmol per 1 μg of BSM) were identified with 14 major (>1.0%), 12 minor (0.1%-1.0%), and eight trace (<0.1%)
-glycans, which were characterized based on their constituents (sialylation (14
-glycans; 81.9%, sum of relative quantities of each glycan), non-sialylation (20; 18.1%), fucosylation (20; 17.5%), and terminal-galactosylation (6; 3.6%)) and six core structure types Gal-GalNAc, Gal-(GlcNAc)GalNAc, GlcNAc-GalNAc, GlcNAc-(GlcNAc)GalNAc, and GalNAc-GalNAc.
-glycosylation sites were identified using
-glycopeptides (bold underlined;
GE
R
VI,
H
GR
R
I,
G
P
V
AEQI,
RP
YGAL,
Q
LGPL,
M
R
VVV, and
RPEDN
AVA) obtained from proteolytic BSM; these sites are in the four domains of BSM. The gel-forming mucins share common domain structures and glycosylation patterns; these results could provide useful information on mucin-type
-glycans. This is the first study to characterize
-glycans and identify
-glycosylation sites in BSM.
Bovine submaxillary mucin (BSM) is a heavily-glycosylated macromolecular (approximately 4 MDa) protein and is used in various biomaterial applications in light of its high viscosity and ...biocompatibility, in addition to use as a biochemical substrate or inhibitor as a result of its abundant O-glycans. Although it has been reported that N-glycosylation provides stability of human mucins, most BSM research has been focused on its O-glycans, while N-glycans have not been reported to date. In this study, a common N-glycan core component was detected by monosaccharide analysis of BSM, and the structures of the N-glycans and their relative quantities were determined by liquid chromatography–tandem mass spectrometry. Seventeen N-glycans comprising ten complex-type Fucose0~2Hexose3~4N-acetylhexosamine1~6Sulfate0~1; 61.1% (the sum of the relative quantities of each N-glycan out of the total N-glycans), two high-mannose-type (Hexose5~6N-acetylhexosamine2; 12.0%), and five paucimannose type (Fucose0~1Hexose3~4N-acetylhexosamine2~3; 26.9%) were identified, but no hybrid-type or sialylated N-glycans were found. Additionally, these are less-branched structures compared to human mucins. Of these, ten glycans (77.2%), including two sulfated glycans (8.0%), were core fucosylated, which confer unique biological functions to glycoproteins. The N-glycosylation sites were identified from the analysis of glycopeptides from BSM. This study is the first confirmation of N-glycan attachment to BSM.
•BSM is used in various biomaterial applications and as a biochemical reagent.•BSM contains abundant O-glycans, but N-glycans have not been reported to date.•This study characterized 17 N-glycans and their relative quantities in BSM.•BSM contains core-fucosylated and sulfated N-glycans but not sialylated glycans.•BSM N-glycans are less-branched structures compared to human mucins.
Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we ...identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (−10.9% and −18.6%, respectively), while bi- and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.
•Protein-specific glycoengineered CHO cells were constructed.•Overexpression of GnT-IV played a major role in antennary structure formation of CTLA4-Ig.•Co-overexpression of GnT-IV and α2,6-ST made highly sialylated CTLA4-Ig.
•Plant core-fucosylated glycans may elicit an immune response in humans.•Rice cell-derived rhGAAs were produced under eight different culture conditions.•N-glycans of eight rhGAAs were analyzed using ...UPLC and LC-ESI-HCD-MS/MS.•15 N-glycans were characterized as core-, de-core-, and non-core-fucosylated glycans.•The lowest core-fucosylated condition of rhGAA was 2-FF combined with PF-68.
Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 μM of 2-fluoro-l-fucose (2-FF), 50 μM of 2-FF, 100 μM of 2-FF, 100 μM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 μM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 μM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.
The benefits of intraoperative magnesium supplementation have been reported. In this prospective, randomized study, the effects of magnesium supplementation during general anaesthesia on emergence ...delirium and postoperative pain in children were evaluated.
A total of 66 children aged 2 to 5 years who underwent strabismus surgery were assigned to the magnesium or to the control group. Preoperative anxiety was assessed using the modified Yale Preoperative Anxiety Scale. After anaesthesia induction, the magnesium group received an initial loading dose of 30 mg/kg magnesium sulphate over 10 min and, then, continuous infusion of 10 mg/kg per h until 10 min before the end of the surgery. The control group received an equal volume of normal saline via the same regimen. The Paediatric Anaesthesia Emergence Delirium (PAED) score, pain score, and respiratory events were assessed at the postanaesthetic care unit.
Data obtained from 65 children were analyzed. The PAED and pain scores of the two groups did not differ significantly. There were 26 of 33 (78.8%) and 27 of 32 (84.4%) children with emergence delirium in the control and the magnesium groups, respectively (odds ratio 0.69, 95% CI 0.19-2.44; p = 0.561). The preoperative anxiety score was not significantly correlated with the PAED score. The incidence of respiratory events during the emergence period did not differ significantly between the two groups.
Magnesium supplementation during anaesthesia had no significant effects on the incidence of emergence delirium or postoperative pain in children undergoing strabismus surgery.
ClinicalTrials.gov ( NCT03132701 ). Prospectively registered May 8, 2017.
A beam position monitor with high precision and resolution is required to control the beam trajectory for matching to the injection orbit and acceleration in a heavy-ion synchrotron. It will be also ...used for measuring the beta function, tune, and chromaticity. Since the bunch length at heavy ion synchrotron is relatively long, a few meters, a boxlike device with plates of typically 20cm length is used to enhance the signal strength and to get a precise linear dependence with respect to the beam displacement. Especially, the linear-cut beam position monitor is adopted to satisfy the position resolution of 100µm and accuracy of 200µm for a nominal beam intensity in the KHIMA synchrotron of ∼7×108 particles for the carbon beams and ∼2×1010 for the proton beams. In this paper, we show the electromagnetic design of the electrode and surroundings to satisfy the resolution of 100µm, the criteria for mechanical aspect to satisfy the position accuracy of 200µm, the measurement results by using wire test-bench, design and measurement of a high input impedance pre-amplifier, and the beam-test results with long (∼1.6μs) electron beam in Pohang accelerator laboratory (PAL).
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