CDR2L Antibodies: A New Player in Paraneoplastic Cerebellar Degeneration

<div><p>Objective</p><p>Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens.</p><p>Methods</p><p>Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins.</p><p>Results</p><p>RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane.</p><p>Interpretation</p><p>Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD.</p></div

Three patient sera with different Yo antibody composition are shown.

<p>The first serum reacts with CDR2 (A1–3), but not CDR2L (A4–6). The second serum reacts with CDR2L (B4–6), but not CDR2 (B1–3). The third serum reacts with both CDR2 (C1–3) and CDR2L (C4–6). The first row shows HeLa cells transfected with CDR2 fused with GFP (A1, B1, C1) or CDR2L fused with GFP (A4, B4, C4) in green. The second row shows the immunocytochemical labelling of the transfected cells with patient serum and a secondary antibody goat-anti-human Alex Fluor 594 in red. The third row shows the overlay of GFP fluorescence and antibody-labelling, co-localization appears in yellow. Note that the first serum shows strong co-localization with CDR2 (A3), but not CDR2L (A6), the second serum shows strong co-localization with CDR2L (B6), but not CDR2 (B3), and the third serum shows strong co-localization with both CDR2 (C3) and CDR2L (C6). The results show that Yo sera can contain CDR2, CDR2L or both antibodies. Scale bar 25 µm.</p

Yo positive serum containing CDR2L stains Purkinje cells.

<p>Serum from a patient with PCD and Yo antibodies (both anti-CDR2 and anti-CDR2L) shows granular cytoplasmic staining of Purkinje cells in rat cerebellar section (A). Similar staining of the serum was observed after absorption with recombinant CDR2 (B). However, no staining was observed when the serum was absorbed with recombinant CDR2L (C). Scale bar 10 µm.</p

Staining of human Purkinje cells with CDR2 and CDR2L antibodies.

<p>Granular cytoplasmic reaction of human Purkinje cells with polyclonal antibody against the C-terminal (A) and the N-terminal (B) of the CDR2L protein. The C-terminal CDR2 antibody also gives some staining of the molecular and granular layers of the cerebellum (A). The polyclonal antibody against CDR2 did not stain the cerebellar sections (C). Scale bars 50 µm.</p

Patients with both CDR2 and CDR2L antibodies.

<p>P. cell = Purkinje cell staining.</p><p>IP = Immunoprecipitation.</p><p>IF = Immunofluorescence.</p><p>LB = Line blot.</p><p>PNS = Paraneoplastic neurological syndrome.</p><p>PCD = paraneoplastic cerebellar degeneration.</p

Localization of CDR2L and CDR2 in HeLa cells.

<p>HeLa cells transfected with CDR2L-myc (A1–6) or CDR2-myc (B1–6) were immunocytochemically stained with anti-myc antibodies (green, A2, A5, B2, B5). The membrane was visualized with wheat germ agglutinin (WGA, red, A1, A4, B1, B4). This was performed as a surface staining without addition of Triton-x-100 (A1–3, B1–3) or with Triton-x-100 to permeabilise the membrane (A4–6, B4–6). To show the membrane localisation of CDR2L, only selected slices of a z-stack were projected on top of each other, namely 16 (A1–3) or 23 (A4–6) slices. To show that CDR2 is not localised to the membrane, all slices covering the cells were superimposed, namely 55 (B1–3) and 40 (B4–6) slices. The overlays show that CDR2L can be detected in the membrane (A3), but not CDR2 (B3). Staining of non-permeabilised cells in B2 shows that CDR2 is not membrane-bound. However, the cells were successfully transfected with CDR2 as shown when the cells were permeabilised (B5). The results show that in the permeabilised cells, CDR2L (A5) is localized to the cytoplasm and cellular membrane, whereas CDR2 (B5) is only localised to the cytoplasm and nucleus. Scale bars 20 µm.</p