High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Identification and characterization of CEPU-Se-A secreted isoform of the IgLON family protein, CEPU-1.

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Identification of chURP, a nuclear calmodulin-binding protein related to hnRNP-U.

In a screen for myosin-like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP-U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U-related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP-U, although chURP and hnRNP-U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin-binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.