Expression of candidates in HEK 293T cells and binding assay

From our proteomic analysis of glycoprotein-enriched and non-compact myelin fractions we generated a list of candidate proteins by transmembrane and signal peptide prediction. We then created a soluble version of each candidate, cloning the signal peptide and predicted extracelular domain and tagging it with a human Fc fragment for detection. The exclusion of the transmembrane domain thus results in the protein being secreted by transfected HEK 293T cells, and the soluble proteins were then retrieved by collecting the supernatant.

Subsequently, a binding assay was used as a screening method to detect potential inter-action of selected candidates with neurons or oligodendrocytes (as some of the axonal components may be isolated along with myelin). The assay was performed by adding the soluble version of the candidate proteins to neurons and oligodendendrocyte-enriched cultures. As positive controls, we used Necl1-Fc and Necl4-Fc, which bind strongly to oligodendrocytes and axons, respectively (Spiegel et al., 2007).

We evaluated the expression and secretion of Necl1, Necl4 and candidate proteins by analysis of the supernatant of transfected HEK cells by western blot (see example in Figure 3.30).

Figure 3.30: Western blot of Fc-fused candidate proteins. pcDNA is the empty vector, used as negative control. The soluble proteins consisted of the signal peptide and extracellular domain, fused to a human Fc fragment for detection. The proteins are retrieved from the supernatant of transfected HEK 293T cells and detected using an antibody against human Fc fragment. Necl: Nectin-like protein, Lsamp:

limbic system-associated membrane protein, Opcml: opioid binding protein/cell adhesion molecule-like, Ntm:

neurotrimin, MCAM: melanoma cell adhesion molecule, Plekhb1: pleckstrin homology domain containing, family B (evectins) member 1: LGI3: leucine-rich repeat LGI family, member 3.

To evaluate whether the expressed soluble proteins retained their normal interaction prop-erties, we took the supernatant from transfected HEK 293T cells, incubated it with Cy3-conjugated anti-Fc antibody and added it to live cells for 20 minutes. Figure 3.31 shows a sample of a binding assay using Necl1 and Necl4 as controls.Using this binding assay as a screening tool, we screened for the candidates as Igsf8, MCAM, Plekhb1 and LGI3, and did not observe binding (see example for Igsf8 in Figure 3.32).

We decided to take MCAM-Fc as a negative control for the functional assays, to discard unspecific effect of a soluble Fc-fusion protein.However, we saw binding of one of our can-didates, limbic system-associated membrane protein to both neurons and oligodendrocytes (Lsamp,Figure 3.33).

The Lsamp-Fc fusion protein did not appear to bind to astrocytes. Uptake by microglia was observed, but was considered unspecific as it occured with all proteins that were added microglia and can, thus, be attributed to its phagocytic properties (Figure 3.34). This suggests that the binding to oligodendrocytes and neurons observed is specific.

Figure 3.31: Binding assay of Necl1-Fc and Necl4-Fc as controls. Soluble versions of Necl1 and Necl4, consisting of the extracellular domain fused to a human Fc fragment were tagged with Cy3-conjugated anti- Fc antibodies and added to neuronal and oligodendrocyte cultures. As described previously, Necl1-Fc binds extensively to oligodendrocytes, stained with MBP, whilst Necl4-Fc binds to neurons, stained withβIII tubulin (Spiegel et al., 2007). Scale bar: 50µm.

Figure 3.32: Immunoglobulin family member 8 (Igsf8) binding assay. Binding assay of Igsf8 to neurons and oligodendrocytes,using Necl1-Fc and Necl4-Fc as controls. Soluble Fc-fusion proteins were added to neuronal and oligodendrocyte cultures. No binding of Igsf8 was observed to oligodendrocytes (MBP) nor neurons (βIII tubulin). Transfected HEK 293T cells were stained with Cy3-conjugated anti-Fc antibodies to reveal the expression of the proteins within the cell. Scale bar: 50µm.

Figure 3.33: Limbic system-associated membrane protein (Lsamp) binding assay. Binding assay of Lsamp to primary neuronal and oligodendrocyte culture, using Necl1-Fc and Necl4-Fc as controls.

Soluble versions of the proteins, consisting of the extracellular domain fused to a human Fc fragment were tagged with Cy3-conjugated anti-Fc antibodies and added to neuronal and oligodendrocyte cultures. Positive binding of Lsamp to both oligodendrocytes (MBP) and neurons (βIII tubulin) was observed. Scale bar: 50 µm

Figure 3.34:Lsamp does not bind to astrocytes and is uptaken by microglia.Binding assay of Lsamp to primary astrocytes and microglia culture present in primary neuronal cultures,using MCAM-Fc as control. No binding to astrocytes (GFAP) was observed. Microglia uptake of both Lsamp-Fc and MCAM-Fc is shown. Scale bar: 50µm

Both candidate proteins Lsamp and Neurotrimin (here termed Ntm) belong to the GPI-anchored family IgLON, and Lsamp has been previously identified in other myelin pro-teomic and transcriptomic analysis (Nielsen et al., 2006; Jahn et al., 2009a). We therefore decided to include a third member of the family Opcml, to analyze whether the binding property is shared among the family. Opcml was found in the proteomic screening but failed to pass the first transmembrane prediction by TMHMM and Phobius and was not initially included in the candidate list.

However, as the IgLON family is GPI-anchored, they are anchored to the membrane and can be involved in cellular communication as they can associate to transmembrane proteins to trigger intracellular responses (Simons and Toomre, 2000). Lsamp, Opcml and Ntm have previously been described as neuronal proteins that appear to regulate neurite growth and interact with members of the same family, forming both homophilic and heterophilic complexes (Lodge et al., 2000; Gil et al., 2002; Reed et al., 2004). Concordantly, we observed that the three proteins bound to neurons (Figure3.35).

Figure 3.35: Neuronal binding assay with proteins of the IgLON family.Proteins of the IgLON family, Lsamp, Opcml and Ntm were used for binding assays with primary neuronal culture,using MCAM-Fc as negative control. Soluble versions of the proteins, consisting of the extracellular domain fused to a human Fc fragment were tagged with Cy3-conjugated anti-Fc antibodies and added to neuronal and oligodendrocyte cultures. Positive binding of Lsamp, Opcml and Ntm to neurons (βIII tubulin) was observed. Scale bar: 50 µm

The confirmation of the interaction of IgLON candidates with neurons primary culture shows that our fusion proteins were biologically functional. We therefore proceeded to repeat the binding assay with Lsamp, Opcml and Ntm in oligodendrocytes, using MCAM-Fc as a negative control. To protect the integrity of the oligodendrocyte lipid-rich myelin sheets for our binding assay, we did a surface staining against the oligodendrocyte marker O1, which requires no permeabilization. We saw binding of Lsamp, Opcml and Ntml (Figure 3.36).

Figure 3.36: Oligodendrocyte binding assay with proteins of the IgLON family. Proteins of the IgLON family, Lsamp, Opcml and Ntm were used for binding assays with primary oligodendroglial culture,using MCAM-Fc as negative control. Soluble versions of the proteins, consisting of the extracellular domain fused to a human Fc fragment were tagged with Cy3-conjugated anti-Fc antibodies and added to neuronal and oligodendrocyte cultures. Positive binding of Lsamp, Opcml and Ntm to oligodendrocytes (MBP) was observed. Scale bar: 50µm

To confirm that the IgLON proteins are interacting with other members of their family, we transfected HEK 293T cells with full length Lsamp tagged with EGFP. Then we added the soluble proteins and visualized them with a Cy3-conjugated Fc antibody. We indeed observe how there is interaction of Lsamp with the other members of the family, as well as itself (Figure3.37). No binding was observed adding MCAM-Fc or adding the IgLON

proteins to HEK 293T cells transfected only with EGFP, confirming the specificity of the interaction. Similar results where observed when expressing the full length of Opcml and Ntm (data not shown).

Figure 3.37: IgLON fusion proteins interact with members of their family. Lsamp-Fc, Opcml-Fc and Ntm-Opcml-Fc were added to HEK 293T cells transfected with full length Lsamp-EGFP, using MCAM as negative control. IgLON proteins bound to HEK 293T cells expressing full length Lsamp but did not bind to HEK cells transfected with EGFP. Scale bar 100µm

To test the specificity of commercially available antibodies we transfected HEK 293T cells with myc-tagged full length proteins, and tested different shRNA to knockdown the expression of the proteins, using scramble shRNA and untransfected HEK 293T cells as negative controls. Unfortunately, the antibodies commercially available have not been useful in elucidating the expression of the IgLON candidates the different cell types, as they seem to give unspecific signals which do not facilitate the expression analysis (Figure 3.38).

To analyze the distribution of exogenously expressed Lsamp, Opcml and Ntm in oligoden-drocytes, we transiently transfected primary oligodendrocytes with full-length version of these proteins, with a myc tag in the N-terminus. We observed that the proteins entered the compact, MBP positive myelin-like sheets (Figure 3.39).

Figure 3.38:Knockdown of exogenously expressed IgLON proteins in HEK 293T cells.HEK 293T cells were transfected with myc-tagged full length Lsamp, Opcml and Ntm. Knockdown was attempted using four shRNA constructs, a scramble shRNA as control. Knockdown was assessed staining against myc, and commercially available antibodies for Lsamp (Biozol), Opcml (Biozol) and Ntm (Millipore) were used to validate the specificity of their signal. Only Ntm seemed to reflect the pattern obtained by myc antibodies, but presents a stong unspecific band at approximately 50 KDa.

Figure 3.39: Exogenous expression of IgLON proteins in oligodendrocytes. Myc-tagged full length Lsamp, Opcml and Ntm localized to MBP-positive myelin sheets in mature oligodendrocytes in vitro.

Scale bar: 50µm.