1. Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein Akinori Kashimura., Kazuaki Okawa., Kotarou Ishikawa., Yuta Kida, Kokoro Iwabuchi, Yudai Matsushima, Masayoshi Sakaguchi, Yasusato Sugahara, Fumitaka Oyama* Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo, Japan Speaker: 黃振哲 Advisor: 林富邦 博士

2. Abstract Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His) 6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A- AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β- D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4- nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'- diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.

3. AIM E. coli expression CHO expression AMCase is similarity

4. introduction Chitin Chitin is a polymer of (β-1.4)-linked N-acetyl-D-glucosamine Chitin is a composition of 1. microfilarial sheaths of parasites 2. cell walls in fungus 3. exoskeleton of crustaceans and insects

5. Chitinase Chitinase (EC 3.2.1.14) hydrolyzes chitin Chitin metabolism in a wide range of organisms 1. bacteria 2. fungi 3. nematodes 4. arthropod

6. Mammals chitinases Mammals has two active chitinases 1. chitotriosidase (Chit1) 2. acidic mammalian chitinase (AMCase) The sequence is homology with bacterial chitinases and belong to the family 18 of glycoside hydrolases (Henrissat B 1991)

7. chitotriosidase (Chit1) Chit1 was the first mammalian chitinase to be purified and cloned (Renkema et.al 1995) The physiological role is defense against to chitin-containing pathogens Recessively inherited deficiency(Chit1) in Caucasians, suggesting that Chit1 as a sole chitinase, performing a defensive function under normal circumstances Marked elevation of Chit1 activity in Gaucher disease(lysosomal storage disorder)

8. Acidic mammalian chitinase (AMCase) was named for its acidic isoelectric point (Boot RG, et al. 2001) is a 50 kDa and is expressed in the mouse stomach and lung can withstand a low pH environment has been shown to be most active at pH 2.0 and is acid-stable AMCase

9. Research the causes of AMCase Pathological conditions Induced asthma mouse model Antigen-induced mouse models of allergic lung inflammation (Reese TA, et al. 2007) In humans, AMCase { } are associated with bronchial asthma AMCase expression ↑

10. Recently, in the mouse stomach, AMCase mRNA is synthesized = pepsinogen C (Ohno M, et al. 2013) AMCase may play an important role in asthma, immune response and food digestion. Little is known, about the pathophysiological functions of AMCase in mice and humans.

11. In this study Target gene expression in these system 1. mammalian cell 2. insect cell expression system 3. E. coli We describe an E. coli expression system (Lowenadler B, et al. 1987) Protein A active AMCase fused to V5 epitope → Protein A-AMCase-V5-His (His) 6 tag

12. E. coli-produced fusion protein that have two activities 1. chitinolytic 2. chitin-binding activities Both two activities were comparable to mammalian cultured cell- expressed AMCase

13. Use pEZZ18 expression vector 1. staphylococcus aureus Protein A promoter 2. signal sequence 3. truncated form of Protein A (synthetic ZZ domain) 4. pEMBL8 +

14. Materials and Methods Mammalian Expression Vector Mouse Total RNA Master Panel → mouse stomach total RNA cDNA Forward primer : EcoRI-pre-AMCase 5’-CATGGAATTCCGGGAGGAACGATGGCCAAGCTACT-3’ Reverse primer : XhoI-pre-AMCase 5’-GTGACCTCGAGCTGGCCAGTTGCAGCAATTACAGC-3’ (Ohno M, et al. 2012)

15. Product purify : Wizard SV Gel and PCR Clean-Up System Subclone : pcDNA3.1/V5-His C vector (similarly digeste) Sequence : pcDNA3.1/pre-AMCase-V5-His

16. AMCase cDNA

17. E. coli Expression Vector Template : pcDNA3.1/pre-AMCase-V5-His Forward primer : EcoRI-mature-AMCase 5’-CATGGAATTCGTACAATCTGATATGCTATTTCACC-3’ Reverse primer : SalI-pcDNA BGH 5’-AGGGGTCGACTAGAAGGCACAGTCGAGGCTGATCA-3’

18. Subclone : pEZZ18 (similarly digeste) Plasmid DNA : pEZZ18/pre-Protein A-AMCase-V5-His Control : pre-Protein A-V5-His

19. The Preparation of the Recombinant Protein from the Medium, Periplasmic Space and Soluble Fractions of E. coli 5,000 x g, 20 min, 4 ℃

20. The supernatants were combined (periplasmic space 1/osmotic shock fraction)

21. periplasmic space 2/lysozyme fraction

22. cytoplasmic soluble fraction

23. periplasmic space 1 periplasmic space 2 cytoplasmic soluble crude

24. The Separation of the Recombinant Protein from the Insoluble Fractions by Ni Sepharose solubilized ‘‘insoluble fraction’’

25. Wash (column) : 8 M urea (10-column volumes ) Wash (resin) : 0.05 M imidazole, 0.5 M NaCl (10-column volumes ) Elute (bind proteins) : 0.5 M imidazole, 0.5 M NaCl desalted

26. The Transient Expression in CHO Cells and the Purification of AMCase-V5-His from CHO Culture Medium

27. Determination of Protein Concentration, SDS polyacrylamide Gel Electrophoresis and Western Blotting periplasmic space 1 periplasmic space 2 cytoplasmic soluble solubilized ‘‘insoluble fraction’’ Bradford ProteinSDS-PAGE Staining Coomassie Blue R-250 Electrophoretically transfer Results

28. incubate anti-V5-HRP Monoclonal antibody Substrate Immobilon Western Chemiluminescent HRP Luminescent Image Analyzer Results

29. Result 1

30. Chitinase Enzymatic Assays periplasmic space 1cytoplasmic soluble periplasmic space 2 solubilized ‘‘insoluble fraction’’ Substrate, 200 µm 4-Nitrophenyl N,N′- diacetyl-β-D-chitobioside At optimum pH and temperature Buffer : McIlvaine’s buffer 0.1 M Gly-HCl buffer 50µL : E. coli- or CHO-expressed protein

31. 37 ℃, 30 min optimum pH kinetic assays Reactions were halted Measure absorbance 4-nitrophenolate ion

32. Result 2

33. The Effects of pH and Temperature on Chitinase Activity Optimal pH : Substrate : 4-Nitrophenyl N,N′-diacetyl-β-D-chitobioside Buffer : McIlvaine’s buffer (pH 2.0 ~ 8.0) 0.1 M Gly-HCl buffer (pH 1.0 ~ 4.0) Condition : 37 ℃, 30min pH preference : recombinant AMCase = native enzyme from the mouse intestine

34. Optimal temperature Substrate : 4-Nitrophenyl N,N′-diacetyl-β-D-chitobioside Buffer : 0.1 M Gly-HCl buffer (pH 2.0) Temperature range : 30 ℃ ~ 60 ℃ Reation time : 15min

35. pH stability Samples were incubated in buffer (on ice, 1H) Buffer : 1. 0.1 M Gly-HCl buffer (pH 1.0 to pH 4.0) 2. McIlvaine’s buffer (pH 2.0 to pH 8.0) 3. Clark and Lubs buffer (0.1 M KCl, 0.1 M H 3 BO 3 and 0.1 M NaOH; pH 8.0 to pH 10.0) 4. Carbonate buffer (0.05 M NaHCO3 and 0.1 M NaOH; pH 10.0 to pH 11.0) E. coli-expressed AMCase exhibited robust stability under basic as well as acidic conditions

36. Heat stability Samples were incubated in McIlvaine’s buffer in 0.1 M Gly-HCl buffer (pH 2.0 or 7.0) Condition : 30 ℃ ~ 58 ℃, 20 min After pre-incubation : Measured the 4-Nitrophenyl N,N′-diacetyl-β-D-chitobioside Indicated : Recombinant AMCase is heat stable both in acidic and neutral conditions.

37. Chitin Binding Assay Wash three times CHO-expressed AMCase-V5-His E. coli-expressed Protein A-AMCase-V5-His Protein A-V5-His (control protein) [0.5 M NaCl in 20 mM Tris- HCl (pH 7.6)]

38. Mix in ice, 1h 2,000 x g, 5 min unbound supernatantWestern blot bind five timesMix

39. 95 ℃, 5min Western blot

40. Result 3

41. The Degradation of Colloidal Chitin by E. coli- or CHO expressed Mouse AMCase Colloidal chitin (final concentration of 1 mg/mL) E. Coli expressed (50µl) 0.1 M Gly-HCl buffer (pH 2.0) CHO expressed (50µl) 0.1 M Gly-HCl buffer (pH 2.0) 37 ℃, 1h (keep)By AMCase proteins Note : Standard - N-acetyl chitooligoaccharides Fluorophore with reducing end groups

42. isolation Result

43. Taken together, these results indicate that E. coli-expressed AMCase can be considered to be a functional enzyme comparable to CHO expressed AMCase Result 4

44. Evaluate chitin hydrolytic activities AMCase 4-Nitrophenyl N,N′- diacetyl-β-D-chitobioside CHO E. coli Western blot

45. CHO-expressed AMCase and E. coli-produced AMCase gave similar signals in the immunoblot analysis Result 5

46. E. Coli Native Optimal pH and acid stability = native chitinase Recombinant AMCase facilitates chitin binding Recombinant AMCase N,N′-diacetyl-chitobioside E. coli-expressed AMCase properties = AMCase compare degrade colloidal chitin Native enzyme CHO-expressed AMCase Discussion

47. Paper contributed Mouse AMCase may play important physiological roles in nutrition A genetic variant in the human AMCase gene modulate the enzymatic activity In COS-7 cells, site-directed mutagenesis of AMCase expressed His187 is responsible for the acidic optimum pH For elucidating biological functions of the enzyme and performing a detailed structure and functional relationship analysis E. coli-produced recombinant protein

48. Inhibition of AMCase therapeutic strategy of asthma In this study, use the recombinant AMCase substrate specificity analysis of the product medical interest

49. Thank you

50. Isoelectric focusing profile of chitinolytic activity in mouse lung extract pI : 4.5 5.5-6.5 4MU-chitotrioside substrate (chitin-like 4-methylumbelliferyl- b-chito-oligosaccharide )

51. , purified human recombinant chitotriosidase, purified mouse AMCase The mouse acidic chitinase shows a pronounced pH optimum at pH 2.3 Chitotriosidase inactivated at low pH

52. The fluorophore-assisted carbohydrate electrophoresis technique was used to visualize the cleavage products of recombinant human chitotriosidase and recombinant mouse AMCase using colloidal chitin as substrate. Lane 1, no enzyme added. Lane 2, products formed after incubation with 50- kDa recombinant human chitotriosidase and chitin. Lane 3, products formed with recombinant mouse AMCase and chitin. Lane 4, human chitotriosidase incubated without substrate. Lane 5, mouse AMCase incubated without substrate.

53. Mouse AMCase cDNA sequence and deduced amino acid sequence cDNA sequence (GenBankTM accession number AF290003) The hydrophobic signal peptide (amino acids 1–21) is underlined with a single line The putative chitin binding domain (amino acids 426–473) is underlined with a double line The hinge region separating the catalytic domain from the chitin binding domain is underlined with a dashed line The part of the protein purified from mouse intestine that was determined by Edman sequencing is boxed

54. Protein A gene fusion vector system Based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus Protein A secretory proteins, short proteins (extracellular expression ) Staphylococcus aureus Protein A promoter control, not pEZZ18 contains a signal sequence → secreted into aqueous culture medium In our case, most of AMCase was present in periplasmic fraction of E. coli use

55. pEZZ18 system is fit for the mouse AMCase If the fusion product is stable under these conditions, this method can only be used Our results, the pEZZ18 system is the best fit for the expression of mouse AMCase, which is an acid-stable secretory enzyme At pH 1 ~ 3 one-step to recovery Eluted : 0.1 M Gly-HCl (pH 2.5)

56. Protein A Protein A(17.8 kDa) has unique folding properties → little effect of folding of protein Because E. coli-express = native enzyme or CHO-express In periplasmic space(E. coli) form an tertiary structure = naturally synthesized Our results, primary structure of AMCase tertiary structure Speculation : ancient chitinase family or periplasmic expression conserved sequence form

58. E. coli Expression Vector Template : pcDNA3.1/pre-AMCase-V5-His Forward primer : EcoRI-mature-AMCase (temple : AMCase cDNA) 5’-CATGGAATTCGTACAATCTGATATGCTATTTCACC-3’ Reverse primer : SalI-pcDNA BGH (temple : pcDNA3.1/V5-His C) 5’-AGGGGTCGACTAGAAGGCACAGTCGAGGCTGATCA-3’