Protein Engineering of ICAM-1 D1 for Biotherapeutics

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ICAM-1

The Intercellular adhesion molecule-1 (ICAM-1, CD54) is a type I transmembrane glycoprotein that plays an important role in the immune and inflammatory response. ICAM-1 is a molecule with many functions but its main functions that interests us are its antigen-independent adhesion between lymphocytes and their targets and its role in migration of leukocytes to the sites of inflammation. Also its role as human rhinovirus receptor is our main interest.


Rhinovirus Therapeutics ICAM-1 consists of five extracellular domains, a transmembrane domain and cytoplasmic domain. In a randomized clinical trial, the efficacy and safety of intranasal administration of a recombinant soluble ICAM-1 D1-D5 has been tested to show that a total of 4.4 mg/d of the molecule was needed for an active treatment. Rhinovirus modulate the two distinct mRNA transcripts coding for ICAM in bronchial epithelial cells with subsequent ICAM-1 expression on the cell surface and down regulation of soluble ICAM-1 release which promote epithelial cell infectivity. Although very low in resolution, an electron micrsocopy reconstructions of complexes D1D2 ICAM with HRV16 and HRV 14 demonstrates that residues important for binding ICAM-1 with rhinovirus has been determined to be located at Domain 1.

Integrin design associated biotherapeutics Lymphocyte function associated antigen-1 is an integrin, noncovalently associated alphabeta heterodimeric transmembrane molecules that mediate cell-cell and cell-extracellular matrix adhesion. ICAM-1D1 also has its ligand binding sites for LFA-1. Because of the key role of the interaction between LFA-1 and ICAM-1 in immune responses, defining its structural basis is of great interest. If a crystal structure of functional, high affinity ICAM-1 D1 and LFA-1 Idomain can be achieved, it can be used for further study for inflammatory response mechanisms and drug design.


Fig.1: Summary of ICAM-1 and its function as binding sites for HRV and LFA-1.

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Protein Engineering Unlike other four domains, D1 is not glycosylated. The big advantage of this is that this protein, than, can be expressed with bacterial cells since bacteria are incapable of expressing glycosylated proteins thus more cost-effective protein production is capable.

In order to achieve our goals, a stable ICAM-1 D1 that can function as a good HRV receptor and a stable ICAM-1 D1 that has high affinity with LFA-1 I Domain needs to be expressed and purified. We have already engineered ICAM-1D1 and I domains mutants that shows functional, that binds to each other. Thus optimization of soluble, stable, functional protein is to be developed.


Fig.2: Protein Purification

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References Efficacy of Tremacamra, a Soluble Intercellular Adhesion Molecule 1, for Experimental Rhinovirus Infection. “Ronald B. Turner; Margaret T. Wecker; Gerhardt Pohl; Theodore J. Witek; Eugene McNally; Roger St. George; Birgit Winther; Frederick G. Hayden.” Journal of American Medical Association 281 (1999): 1797-1804.

Song, Gang, et al. “Rational design of intercellular adhesion molecule-1 (ICAM-1) variants for antagonizing integrin lymphocyte function-associated antigen-1-dependent adhesion.” Journal of biological chemistry 281 (2006).

BellaJordi, RossmannGMichael. “Review: Rhinoviruses and Theri ICAM Receptors.” “Journal of Structural Biology”, 1999: 128, 69-74.

ShimaokaMotomu, TakagiJunichi, TimothyASpringe. “Conformational regulation of integrin structure and function.” “Annu. Rev. Biophys. Biomol. Struct. ”, 2003: 31:485 - 516.

Babu, P.V. Cherish, V.K. Srinivas, V. Krishna Mohan, and Krishna Ella. “Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E.Coli.” Journal of Chromatography B, 2008: 218-226.

Batas, boris, Chiara Schiraldi, and Julian B Chaudhuri. “Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography.” Journal of Biotechnology, 1998: 149-158.

Nature Publishing Group. Striving for purity: advances in protein purification. 2005. www.nature.com/naturemethods (accessed 8 12, 2008). Wu, Kongtian, et al. “Construction, purification, and immunogenicity of recombinant cystein-cystein type chemokine receptor 5 vaccine.” Protein Expression and urification, 2006: 108-113.

Yang, Yuting, et al. “Structural Basis of Dimerization of ICAM-1 on the cell surface.” Molecular CEll, 2004: Vol.14, 269-276.