1.Expression optimization and molecular modification of heparin C5 epimerase.
Bingbing WANG ; Zhengxiong ZHOU ; Xuerong JIN ; Jianghua LI ; Zhongping SHI ; Zhen KANG
Chinese Journal of Biotechnology 2020;36(7):1450-1458
Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals
;
Carbohydrate Epimerases
;
biosynthesis
;
chemistry
;
genetics
;
Escherichia coli
;
Gene Expression
;
Heparin
;
metabolism
;
Heparitin Sulfate
;
metabolism
;
Iduronic Acid
;
metabolism
;
Zebrafish Proteins
;
biosynthesis
;
chemistry
;
genetics
2.Structural basis of heparan sulfate-specific degradation by heparinase III.
Wei DONG ; Weiqin LU ; Wallace L MCKEEHAN ; Yongde LUO ; Sheng YE
Protein & Cell 2012;3(12):950-961
Heparinase III (HepIII) is a 73-kDa polysaccharide lyase (PL) that degrades the heparan sulfate (HS) polysaccharides at sulfate-rare regions, which are important co-factors for a vast array of functional distinct proteins including the well-characterized antithrombin and the FGF/FGFR signal transduction system. It functions in cleaving metazoan heparan sulfate (HS) and providing carbon, nitrogen and sulfate sources for host microorganisms. It has long been used to deduce the structure of HS and heparin motifs; however, the structure of its own is unknown. Here we report the crystal structure of the HepIII from Bacteroides thetaiotaomicron at a resolution of 1.6 Å. The overall architecture of HepIII belongs to the (α/α)₅ toroid subclass with an N-terminal toroid-like domain and a C-terminal β-sandwich domain. Analysis of this high-resolution structure allows us to identify a potential HS substrate binding site in a tunnel between the two domains. A tetrasaccharide substrate bound model suggests an elimination mechanism in the HS degradation. Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively. The structure of HepIII and the proposed reaction model provide a molecular basis for its potential practical utilization and the mechanism of its eliminative degradation for HS polysaccarides.
Amino Acid Sequence
;
Bacteroides
;
enzymology
;
Catalytic Domain
;
Crystallography, X-Ray
;
Heparitin Sulfate
;
metabolism
;
Kinetics
;
Models, Molecular
;
Molecular Sequence Data
;
Polysaccharide-Lyases
;
chemistry
;
metabolism
;
Substrate Specificity
3.Glycosylation, glycan receptors recognition of SARS-CoV-2 and discoveries of glycan inhibitors against SARS-CoV-2.
Weiyan YU ; Yueqiang XU ; Jianjun LI ; Zhimin LI ; Qi WANG ; Yuguang DU
Chinese Journal of Biotechnology 2022;38(9):3157-3172
COVID-19 represents the most serious public health event in the past few decades of the 21st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.
Antibodies, Neutralizing
;
Glycosylation
;
Heparin
;
Heparitin Sulfate
;
Humans
;
Polysaccharides/chemistry*
;
Receptors, Mitogen/metabolism*
;
SARS-CoV-2
;
Spike Glycoprotein, Coronavirus/metabolism*
;
COVID-19 Drug Treatment
4.The First Korean Case of Mucopolysaccharidosis IIIC (Sanfilippo Syndrome Type C) Confirmed by Biochemical and Molecular Investigation.
Hee Jae HUH ; Ja Young SEO ; Sung Yoon CHO ; Chang Seok KI ; Soo Youn LEE ; Jong Won KIM ; Hyung Doo PARK ; Dong Kyu JIN
Annals of Laboratory Medicine 2013;33(1):75-79
Mucopolysaccharidosis (MPS) III has 4 enzymatically distinct forms (A, B, C, and D), and MPS IIIC, also known as Sanfilippo C syndrome, is an autosomal recessive lysosomal storage disease caused by a deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). Here, we report a case of MPS IIIC that was confirmed by molecular genetic analysis. The patient was a 2-yr-old girl presenting with skeletal deformity, hepatomegaly, and delayed motor development. Urinary excretion of glycosaminoglycan (GAG) was markedly elevated (984.4 mg GAG/g creatinine) compared with the age-specific reference range (<175 mg GAG/g creatinine), and a strong band of heparan sulfate was recognized on performing thin layer chromatography. HGSNAT enzyme activity in leukocytes was 0.7 nmol/17 hr/mg protein, which was significantly lower than the reference range (8.6-32 nmol/17 hr/mg protein). PCR and direct sequencing of the HGSNAT gene showed 2 mutations: c.234+1G>A (IVS2+1G>A) and c.1150C>T (p.Arg384*). To the best of our knowledge, this is the first case of MPS IIIC to be confirmed by clinical, biochemical, and molecular genetic findings in Korea.
Acetyltransferases/*genetics
;
Asian Continental Ancestry Group/*genetics
;
Base Sequence
;
Child, Preschool
;
Chromatography, Thin Layer
;
Female
;
Glycosaminoglycans/urine
;
Heparitin Sulfate/chemistry/metabolism
;
Humans
;
Leukocytes/immunology/metabolism
;
Mucopolysaccharidosis III/*diagnosis/genetics/radiography
;
Mutation
;
Republic of Korea
;
Sequence Analysis, DNA