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1.
J Biotechnol ; 378: 1-10, 2023 Dec 10.
Article En | MEDLINE | ID: mdl-37922995

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.


Antibodies, Monoclonal , Polyamines , Cricetinae , Animals , CHO Cells , Cricetulus , Tunicamycin , Putrescine/metabolism , Eflornithine/pharmacology , RNA, Messenger/metabolism , Glycoproteins , Polysaccharides , Immunoglobulin G , Spermine/metabolism
2.
Sci Rep ; 13(1): 15649, 2023 09 20.
Article En | MEDLINE | ID: mdl-37730951

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.


Craniocerebral Trauma , Hypercholesterolemia , Hyperlipoproteinemia Type II , Animals , Humans , Primates , Hyperlipoproteinemia Type II/genetics , Lipoproteins, LDL , Macaca fascicularis
3.
Chem Pharm Bull (Tokyo) ; 71(7): 616-619, 2023.
Article En | MEDLINE | ID: mdl-37394608

We developed a simple and sensitive analytical HPLC method for the determination of acetylated hyaluronic acid (AcHA) in moisturizing and milk lotions. AcHA with different molecular weights was separated as a single peak using a C4 column and detected through post-column derivatization using 2-cyanoacetamide. The limits of detection and quantification were 60 and 200 ng, respectively. We found that AcHA in water was successfully extracted into a strong anion exchange (SAX) spin column with a recovery rate of AcHA was 63.8 ± 1.8%. Although the supernatant from acetone precipitation of lotions could pass through the spin column, the recovery rate (%) and accuracy of AcHA were affected by the viscous properties of cosmetics and acidic and acetone-soluble ingredients. Upon conducting analytical methods in this study, the concentration of AcHA in nine lotions was found to have ranged from 7.50 to 83.3 µg/mL. These values are comparable to the concentration range of AcHA in emulsions that have been previously evaluated for their superior effects. We believe that the analytical and extraction method is useful for the qualitative analysis of AcHA in moisturizing and milk lotions.


Hyaluronic Acid , Milk , Animals , Chromatography, High Pressure Liquid/methods , Acetone , Emulsions
4.
Carbohydr Polym ; 313: 120847, 2023 Aug 01.
Article En | MEDLINE | ID: mdl-37182947

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1H NMR detected significant signals near 1.2-1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC-MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl-L-fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl-L-fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities.


Chondroitin Sulfates , Pectinidae , Animals , Chondroitin Sulfates/chemistry , Fucose/chemistry , Glycosaminoglycans/chemistry , Heparitin Sulfate , Glucuronic Acid , Glucuronates
5.
Commun Biol ; 5(1): 1071, 2022 10 07.
Article En | MEDLINE | ID: mdl-36207400

Whether a small GTPase RhoA plays a role in the pathology of abdominal aortic aneurysm (AAA) has not been determined. We show here that RhoA expression is reduced in human AAA lesions, compared with normal areas. Furthermore, incidence of AAA formation is increased in vascular smooth muscle cell (VSMC)-specific RhoA conditional knockout (cKO) mice. The contractility of the aortic rings and VSMCs from RhoA cKO mice is reduced, and expression of genes related to the VSMC contractility is attenuated by loss of RhoA. RhoA depletion activates the mitogen-activated protein (MAP) kinase signaling, including MAP4K4, in the aorta and VSMCs. Inhibition of MAP4K4 activity by DMX-5804 decreases AAA formation. Set, a binding protein to active RhoA, functions as an activator of MAP4K4 by sequestering PP2A, an inhibitor of MAP4K4, in the absence of RhoA. In conclusion, RhoA counteracts AAA formation through inhibition of MAP4K4 in cooperation with Set.


Aortic Aneurysm, Abdominal , Muscle, Smooth, Vascular , Protein Serine-Threonine Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/prevention & control , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Mitogens , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , rhoA GTP-Binding Protein/genetics , NF-kappaB-Inducing Kinase
6.
Appl Microbiol Biotechnol ; 106(5-6): 1957-1965, 2022 Mar.
Article En | MEDLINE | ID: mdl-35235007

Arabinoxylan (AX) and arabinoxylooligosaccharides (AXOs) are carbohydrate sources utilized by Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we characterized two genes, BLLJ_1850 and BLLJ_1851, in the hemicellulose-degrading gene cluster (BLLJ_1836-BLLJ_1859) of B. longum subsp. longum JCM 1217. Both recombinant enzymes expressed in Escherichia coli exhibited exo-α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside. BlArafE (encoded by BLLJ_1850) contains the glycoside hydrolase family 43 (GH43), subfamily 22 (GH43_22), and GH43_34 domains. The BlArafE GH43_22 domain was demonstrated to release α1,3-linked Araf from AX, but the function of BlArafE GH43_34 could not be clearly identified in this study. BlArafD (encoded by BLLJ_1851) contains GH43 unclassified subfamily (GH43_UC) and GH43_26 domains. The BlArafD GH43_UC domain showed specificity for α1,2-linked Araf in α1,2- and α1,3-Araf double-substituted structures in AXOs, while BlArafD GH43_26 was shown to hydrolyze α1,5-linked Araf in the arabinan backbone. Co-incubation of BlArafD and BlArafE revealed that these two enzymes sequentially removed α1,2-Araf and α1,3-Araf from double-substituted AXOs in this order. B. longum strain lacking BLLJ_1850-BLLJ_1853 did not grow in the medium containing α1,2/3-Araf double-substituted AXOs, suggesting that BlArafE and BlArafD are important for the assimilation of AX. KEY POINTS: • BlArafD GH43 unclassified subfamily domain is a novel α1,2-L-arabinofuranosidase. • BlArafE GH43 subfamily 22 domain is an α1,3-L-arabinofuranosidase. • BlArafD and BlArafE cooperatively degrade α1,2/3-Araf double-substituted arabinoxylan.


Glycoside Hydrolases , Xylans , Bifidobacterium/enzymology , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Substrate Specificity , Xylans/metabolism
7.
Appl Environ Microbiol ; 88(6): e0218721, 2022 03 22.
Article En | MEDLINE | ID: mdl-35108084

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.


Bifidobacterium longum , Bifidobacterium longum/metabolism , Galactans/metabolism , Glycoside Hydrolases/metabolism , Gum Arabic , Humans , Oligosaccharides/chemistry
8.
J Biochem ; 170(5): 577-585, 2021 Dec 28.
Article En | MEDLINE | ID: mdl-34086898

Transmembrane protein 168 (TMEM168) was found to be localized on the nuclear membrane. A heterozygous mutation (c.1616G>A, p. R539Q) in TMEM168 was identified in patients with Brugada syndrome. This mutation reduced expression of cardiomyocyte sodium channel Nav1.5 via Nedd4-2 E3 ubiquitin ligase-induced ubiquitination and degradation. However, the detailed molecular mechanism provoked by the TMEM168 mutant remains unclear. Here, we demonstrated that small heat shock protein αB-crystallin, which can bind to Nav1.5 and Nedd4-2 and interfere with the association of both proteins, was strongly recruited from the cell surface to the perinuclear region because of the much higher affinity of αB-crystallin with the TMEM168 mutant than with wild-type TMEM168. Following knockdown of αB-crystallin in HL-1 cardiomyocytes, the interaction of Nav1.5 with Nedd4-2 was increased, despite the reduced expression of Nav1.5. Moreover, reduction of Nav1.5 expression by αB-crystallin knockdown was rescued in the presence of a proteasome inhibitor MG-132, suggesting the importance of the αB-crystallin-modulated ubiquitin-proteasome system for the stability of Nav1.5 expression. Collectively, the balance of molecular interactions among Nav1.5, Nedd4-2 and αB-crystallin plays a role in the regulation of cardiomyocyte cell surface expression of Nav1.5, and the TMEM168 mutant disturbs this balance, resulting in a decrease in Nav1.5 expression.


Membrane Proteins/genetics , Mutation , Myocytes, Cardiac/metabolism , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Nedd4 Ubiquitin Protein Ligases/metabolism , alpha-Crystallin B Chain/metabolism , Animals , Brugada Syndrome/genetics , Brugada Syndrome/metabolism , Brugada Syndrome/pathology , Cell Line , Gene Knock-In Techniques , HEK293 Cells , Humans , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/pathology
9.
J Biol Chem ; 296: 100761, 2021.
Article En | MEDLINE | ID: mdl-33971198

Diabetes mellitus (DM) causes injury to tissues and organs, including to the heart and kidney, resulting in increased morbidity and mortality. Thus, novel potential therapeutics are continuously required to minimize DM-related organ damage. We have previously shown that dipeptidyl peptidase III (DPPIII) has beneficial roles in a hypertensive mouse model, but it is unknown whether DPPIII has any effects on DM. In this study, we found that intravenous administration of recombinant DPPIII in diabetic db/db mice for 8 weeks suppressed the DM-induced cardiac diastolic dysfunctions and renal injury without alteration of the blood glucose level. This treatment inhibited inflammatory cell infiltration and fibrosis in the heart and blocked the increase in albuminuria by attenuating the disruption of the glomerular microvasculature and inhibiting the effacement of podocyte foot processes in the kidney. The beneficial role of DPPIII was, at least in part, mediated by the cleavage of a cytotoxic peptide, named Peptide 2, which was increased in db/db mice compared with normal mice. This peptide consisted of nine amino acids, was a digested fragment of complement component 3 (C3), and had an anaphylatoxin-like effect determined by the Miles assay and chemoattractant analysis. The effect was dependent on its interaction with the C3a receptor and protein kinase C-mediated RhoA activation downstream of the receptor in endothelial cells. In conclusion, DPPIII plays a protective role in the heart and kidney in a DM animal model through cleavage of a peptide that is a part of C3.


Diabetic Cardiomyopathies/drug therapy , Diabetic Nephropathies/drug therapy , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/therapeutic use , Heart/drug effects , Kidney/drug effects , Protective Agents/therapeutic use , Animals , Diabetes Mellitus/metabolism , Diabetes Mellitus/physiopathology , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/physiopathology , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/physiopathology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Enzyme Therapy , Heart/physiopathology , Human Umbilical Vein Endothelial Cells , Humans , Kidney/metabolism , Kidney/physiopathology , Male , Mice, Inbred C57BL , Protective Agents/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use
10.
Cancer Res ; 81(9): 2318-2331, 2021 05 01.
Article En | MEDLINE | ID: mdl-33757977

The growth and progression of cancers are crucially regulated by the tumor microenvironment where tumor cells and stromal cells are mutually associated. In this study, we found that stomatin expression was markedly upregulated by the interaction between prostate cancer cells and stromal cells. Stomatin suppressed cancer cell proliferation and enhanced apoptosis in vitro and inhibited xenograft tumor growth in vivo. Stomatin inhibited Akt activation, which is mediated by phosphoinositide-dependent protein kinase 1 (PDPK1). PDPK1 protein stability was maintained by its binding to HSP90. Stomatin interacted with PDPK1 and interfered with the PDPK1-HSP90 complex formation, resulting in decreased PDPK1 expression. Knockdown of stomatin in cancer cells elevated Akt activation and promoted cell increase by promoting the interaction between PDPK1 and HSP90. Clinically, stomatin expression levels were significantly decreased in human prostate cancer samples with high Gleason scores, and lower expression of stomatin was associated with higher recurrence of prostate cancer after the operation. Collectively, these findings demonstrate the tumor-suppressive effect of stromal-induced stomatin on cancer cells. SIGNIFICANCE: These findings reveal that interactions with stromal cells induce expression of stomatin in prostate cancer cells, which suppresses tumor growth via attenuation of the Akt signaling axis.


Cell Proliferation/genetics , Membrane Proteins/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/genetics , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Aged , Animals , Apoptosis/genetics , Cell Communication , Gene Knockdown Techniques , HEK293 Cells , Hep G2 Cells , Humans , Male , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Prostatic Neoplasms/pathology , Stromal Cells/metabolism , Transfection , Tumor Burden/genetics , Xenograft Model Antitumor Assays
11.
FASEB J ; 34(5): 6399-6417, 2020 05.
Article En | MEDLINE | ID: mdl-32175648

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.


Brugada Syndrome/genetics , Genetic Predisposition to Disease , Membrane Proteins/genetics , Mutation , Myocytes, Cardiac/pathology , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Adult , Animals , Brugada Syndrome/pathology , Female , Humans , Male , Membrane Proteins/metabolism , Mice , Myocytes, Cardiac/metabolism , NAV1.5 Voltage-Gated Sodium Channel/genetics , Pedigree , Young Adult
12.
Sci Rep ; 10(1): 188, 2020 01 13.
Article En | MEDLINE | ID: mdl-31932617

Anosmin-1 is a secreted glycoprotein encoded by the ANOS1 gene, and its loss of function causes Kallmann syndrome (KS), which is characterized by anosmia and hypogonadism due to olfactory bulb (OB) dysfunction. However, the physiological function of anosmin-1 remains to be elucidated. In KS, disordered angiogenesis is observed in OB, resulting in its hypoplasia. In this study, we examined the involvement of anosmin-1 in angiogenic processes. Anosmin-1 was detected on the vessel-like structure in OB of chick embryos, and promoted the outgrowth of vascular sprouts as shown by assays of OB tissue culture. Cell migration, proliferation, and tube formation of endothelial cells were induced by treatment with anosmin-1 as well as vascular endothelial growth factor-A (VEGF-A), and further enhanced by treatment with both of them. We newly identified that anosmin-1 activated VEGF receptor-2 (VEGFR2) by binding directly to it, and its downstream signaling molecules, phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC). These results suggest that anosmin-1 plays a key role in the angiogenesis of developing OB through the VEGFR2-PLCγ1-PKC axis by enhancing the VEGF function.


Endothelium, Vascular/cytology , Extracellular Matrix Proteins/metabolism , Neovascularization, Physiologic , Nerve Tissue Proteins/metabolism , Olfactory Bulb/blood supply , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Cell Movement , Cell Proliferation , Cells, Cultured , Chick Embryo , Extracellular Matrix Proteins/genetics , Humans , Morphogenesis , Nerve Tissue Proteins/genetics , Olfactory Bulb/cytology , Olfactory Bulb/metabolism , Receptors, Vascular Endothelial Growth Factor/genetics , Signal Transduction , Vascular Endothelial Growth Factor A/genetics
13.
J Appl Glycosci (1999) ; 67(1): 23-29, 2020.
Article En | MEDLINE | ID: mdl-34429696

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcß1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcß1-Asn-peptides/proteins generated by the action of endo-ß- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

14.
Appl Environ Microbiol ; 85(6)2019 03 15.
Article En | MEDLINE | ID: mdl-30635377

Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside but no activity toward p-nitrophenyl-ß-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan.IMPORTANCE We identified two novel α-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on α1,2/3-l-arabinofuranosyl linkages, while the latter acts on the α1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.


Bacterial Proteins/metabolism , Bifidobacterium longum/enzymology , Glycoside Hydrolases/metabolism , Polysaccharides/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bifidobacterium longum/chemistry , Bifidobacterium longum/genetics , Bifidobacterium longum/growth & development , Cloning, Molecular , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Multigene Family , Sequence Alignment , Substrate Specificity
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