Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins

Abstract

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

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“…Current and future work with the more difficult to produce hydrophobic JAMs (JAM‐B, JAM‐C, and JAM 4) imposes a constrain performing biophysical studies due to the difficulty in purifying them in high yields. Thus, maintaining the MBP fusion will enable high yield and purity of all the recombinant JAMs [16]. In our previous study, MBP E‐CAD displayed constants of affinity very similar to those previously calculated by other biophysical means, thus validating our designs [16].…”

“…Thus, maintaining the MBP fusion will enable high yield and purity of all the recombinant JAMs [16]. In our previous study, MBP E‐CAD displayed constants of affinity very similar to those previously calculated by other biophysical means, thus validating our designs [16]. We also produced, for the first time, chimeric CLDN1 and OCLN that enabled biophysical assays [9].…”

“…Cloning, expression, and purification of targets (JAM‐A, CLDN1, OCLN, and E‐CAD) were prepared as previously described [9,16]. Proteins are cloned in pET28 as C‐terminal to MBP.…”

“…JAMs play roles in vertebrate development and hemostasis [12]. In our previous studies, we presented a strategy based on MBP fusion that enabled us to express and characterize all the members of the family [16]. We reported for the first time that JAMs have both homotypic and heterotypic interactions [16].…”

“…In our previous studies, we presented a strategy based on MBP fusion that enabled us to express and characterize all the members of the family [16]. We reported for the first time that JAMs have both homotypic and heterotypic interactions [16]. To better understand the biophysical properties of JAMs, we prepared epithelial cadherin (E‐CAD) soluble domains fused C‐terminal to MBP.…”