Research on Expression of Somatomedin B Domain of Proteoglycan 4 and Recombinant Protein Aggregation_Journal of Biomedical Engineering

Authors：

WANGLifang ¹ , HANZhibo ² , CHENWenhu ¹ , DUPeng ¹ , SUNAihua ¹ , YANGPing ² ,  ZHAOHongguang ³

1. Department of Basic Medicine, Zhejiang Medical College, Hangzhou 310053, China;
2. State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin 300020, China;
3. Department of Thoracic Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, China;

Corresponding author：

ZHAOHongguang, Email: zhaohg75@126.com

Keywords：

proteoglycan 4; gene expression; structure; protein aggregation

DOI：

10.7507/1001-5515.20140250

Video：

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Abstract Full text Figures/Tables Video References Cited by

Recombinant protein SMB_PRG4 containing two Somatomedin B domains and a small amount of glycosylation of repetitive sequences of proteoglycan 4 was cloned according to PGR4 gene polymorphism. Mature purification process was established and recombinant protein SMB_PRG4, with high-level expression was purified. By using size-exclusion chromatogaraphy and dynamic light scattering, we found that the recombinant protein self-aggregate to dimeric form. Structure prediction and non-reducing electrophoresis revealed that SMB_PRG4 was a non-covalently bonded dimer.

Citation： WANGLifang, HANZhibo, CHENWenhu, DUPeng, SUNAihua, YANGPing, ZHAOHongguang. Research on Expression of Somatomedin B Domain of Proteoglycan 4 and Recombinant Protein Aggregation. Journal of Biomedical Engineering, 2014, 31(6): 1319-1324. doi: 10.7507/1001-5515.20140250 Copy

1.	DENG G, ROYLE G, WANG S J, et al. Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin[J]. J Biol Chem, 1996, 271(22):12716-12723.
2.	ZHUANG P, BLACKBURN M N, PETERSON C B. Characterization of the denaturation and renaturation of human plasma vitronectin. Ⅰ. Biophysical characterization of protein unfolding and multimerization[J]. J Biol Chem, 1996, 271(24):14323-14332.
3.	GIJSBERS R, CEULEMANS H, BOLLEN M. Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1[J]. Biochem J, 2003, 371(Pt 2):321-330.
4.	IKEGAWA S, SANO M, KOSHIZUKA Y, et al. Isolation, characterization and mapping of the mouse and human PRG4(proteoglycan 4) genes[J]. Cytogenet Cell Genet, 2000, 90(3/4):291-297.
5.	JAY G D, TANTRAVAHI U, BRITT D E, et al. Homology of lubricin and superficial zone protein (SZP):products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25[J]. J Orthop Res, 2001, 19(4):677-687.
6.	REES S G, DAVIES J R, TUDOR D, et al. Immunolocalisation and expression of proteoglycan 4(cartilage superficial zone proteoglycan) in tendon[J]. Matrix Biol, 2002, 21(7):593-602.
7.	PANAGOPOULOS I, MERTENS F, ISAKSSON M, et al. Expression of DOL54 is not restricted to myxoid liposarcomas with the FUS-DDIT3 chimera but is found in various sarcomas[J]. Oncol Rep, 2004, 12(1):107-110.
8.	SNAPP K R, CRAIG R, HERRON M, et al. Dimerization of P-selectin glycoprotein ligand-1(PSGL-1) required for optimal recognition of P-selectin[J]. J Cell Biol, 1998, 142(1):263-270.
9.	EPPERSON T K, PATEL K D, MCEVER R P, et al. Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin[J]. J Biol Chem, 2000, 275(11):7839-7853.
10.	AFSHAR-KHARGHAN V, DIZ-KÜÇÜKKAYA R, LUDWIG E H, et al. Human polymorphism of P-selectin glycoprotein ligand 1 attributable to variable numbers of tandem decameric repeats in the mucinlike region[J]. Blood, 2001, 97(10):3306-3307.
11.	LÓPEZ J A, LUDWIG E H, MCCARTHY B J. Polymorphism of human glycoprotein Ⅰb alpha results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. Structure/function implications[J]. J Biol Chem, 1992, 267(14):10055-10061.
12.	KAMIKUBO Y I, OKUMURA Y, LOSKUTOFF D J. Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin[J]. J Biol Chem, 2002, 277(30):27109-27119.
13.	GODL K, JOHANSSON M E, LIDELL M E, et al. The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment[J]. J Biol Chem, 2002, 277(49):47248-47256.
14.	HØGÅSEN K, MOLLNES T E, HARBOE M. Heparin-binding properties of vitronectin are linked to complex formation as illustrated by in vitro polymerization and binding to the terminal complement complex[J]. J Biochem, 1992, 267(32):23076-23082.
15.	STEELE B L, ALVAREZ-VERONESI M C, SCHMIDT T A. Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)[J]. Osteoarthritis Cartilage, 2013, 21(3):498-504.

1. DENG G, ROYLE G, WANG S J, et al. Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin[J]. J Biol Chem, 1996, 271(22):12716-12723.
2. ZHUANG P, BLACKBURN M N, PETERSON C B. Characterization of the denaturation and renaturation of human plasma vitronectin. Ⅰ. Biophysical characterization of protein unfolding and multimerization[J]. J Biol Chem, 1996, 271(24):14323-14332.
3. GIJSBERS R, CEULEMANS H, BOLLEN M. Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1[J]. Biochem J, 2003, 371(Pt 2):321-330.
4. IKEGAWA S, SANO M, KOSHIZUKA Y, et al. Isolation, characterization and mapping of the mouse and human PRG4(proteoglycan 4) genes[J]. Cytogenet Cell Genet, 2000, 90(3/4):291-297.
5. JAY G D, TANTRAVAHI U, BRITT D E, et al. Homology of lubricin and superficial zone protein (SZP):products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25[J]. J Orthop Res, 2001, 19(4):677-687.
6. REES S G, DAVIES J R, TUDOR D, et al. Immunolocalisation and expression of proteoglycan 4(cartilage superficial zone proteoglycan) in tendon[J]. Matrix Biol, 2002, 21(7):593-602.
7. PANAGOPOULOS I, MERTENS F, ISAKSSON M, et al. Expression of DOL54 is not restricted to myxoid liposarcomas with the FUS-DDIT3 chimera but is found in various sarcomas[J]. Oncol Rep, 2004, 12(1):107-110.
8. SNAPP K R, CRAIG R, HERRON M, et al. Dimerization of P-selectin glycoprotein ligand-1(PSGL-1) required for optimal recognition of P-selectin[J]. J Cell Biol, 1998, 142(1):263-270.
9. EPPERSON T K, PATEL K D, MCEVER R P, et al. Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin[J]. J Biol Chem, 2000, 275(11):7839-7853.
10. AFSHAR-KHARGHAN V, DIZ-KÜÇÜKKAYA R, LUDWIG E H, et al. Human polymorphism of P-selectin glycoprotein ligand 1 attributable to variable numbers of tandem decameric repeats in the mucinlike region[J]. Blood, 2001, 97(10):3306-3307.
11. LÓPEZ J A, LUDWIG E H, MCCARTHY B J. Polymorphism of human glycoprotein Ⅰb alpha results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. Structure/function implications[J]. J Biol Chem, 1992, 267(14):10055-10061.
12. KAMIKUBO Y I, OKUMURA Y, LOSKUTOFF D J. Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin[J]. J Biol Chem, 2002, 277(30):27109-27119.
13. GODL K, JOHANSSON M E, LIDELL M E, et al. The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment[J]. J Biol Chem, 2002, 277(49):47248-47256.
14. HØGÅSEN K, MOLLNES T E, HARBOE M. Heparin-binding properties of vitronectin are linked to complex formation as illustrated by in vitro polymerization and binding to the terminal complement complex[J]. J Biochem, 1992, 267(32):23076-23082.
15. STEELE B L, ALVAREZ-VERONESI M C, SCHMIDT T A. Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)[J]. Osteoarthritis Cartilage, 2013, 21(3):498-504.

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