Introduction: Cancerous tumors often induce an inflammatory environment to promote the growth of new vascular tissue. In a typical response to such inflammations, the human immune system uses leukocytes cells to bind to the inflamed endothelial surface [1]. One of the key reactions that leukocytes use to bind to the endothelial cells is the interaction between leukocyte surface integrin protein VLA-4 (very late antigen, alpha4beta1) and endothelial surface protein VCAM-1 (vascular cell adhesion molecule 1)[2, 3]. The tight binding facilitates the cell to move along the endothelial surface and migrate into the subendothelial matrix.

The immune system is able to control this transendothelial migration by changing the integrin affinity state confirmation [4]. As shown in the right image, VLA-4 has three structural states: low affinity, intermediate affinity (not shown), and high affinity[5]. The increased affinity states opens binding sites MIDAS and ADMIDAS on the I domain, allowing for VCAM-1 bonding[6]. The integrin protein constantly shifts between these structures to actively change its binding capability. However the affinity state might possibly be fixed in a certain state by inserting polysaccharide glycan molecules to prevent the protein bending. While the function of the states has been studied, the control of the confirmation continues to be unknown. <box 302px center></box|Figure 1: The low affinity and high affinity structures of Very Late Antigen 4 (VLA-4), image from www.jin-lab.com>

Current Research: I am attempting to fix the VLA-4 proteins in a high confirmation state through the appropriate addition of N-linked glycan groups to specific sites on the integrin protein via mutations. They should act as a stoppers to prevent the VLA-4 protein from bending into a low affinity state. Testing though rolling assays and flow cytometry will help determine the optimal conformation of n-glycogen groups on the integrin protein for binding to VCAM-1.

Future Goals: The long term goal is to establish a cell line with a VLA-4 protein stabilized in a high affinity state. This will allow for the screening of antibodies specific to high affinity VLA-4 protein, creating a powerful new tool for understanding the inflammatory response in vitro and in vivo.

References:

1) Geng, J.G., Directional migration of leukocytes: their pathological roles in infammation and strategies for development of anti-inflammatory therapies. Cell Res, 2001. 11)2_: p85-8

2) Alon R, Kassner PD, Carr MW, Finger EB, Hemler ME, and Springer TA., The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J Cell Biol, 1995. 128(6): p1543-53

3) Berlin C, Bargatze RF, Campbell JJ, von Andrian UH, Szabo MC, Hasslen SR, Nelson RD, Berg EL, Erlandsen SL, and Butcher EC., alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell, 1995. 80(3): p.413-22

4) Luo, B.H., C.V. Carman, and T.A. Springer, Structureal basis of integrin regulation and signaling. Annu Rev Immunol, 2007. 25 p. 619-47 Shimaoka, M. and T.A. Springer, Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discovery, 2003. 2(9): p.703-16

5) Takagi J, Patre BM, Walz T, and Springer T.A., Global conformation rearrangements in integrin extracellular domains in outside-in and inside-out signalling. Cell, 2002. 110(5): p599-611

Nikolai Rakhilin nr233@cornell.edu