Fig. 2. Discovery and validation of a functional transcobalamin receptor autoantibody.

(A) Epitope map of CD320 peptides enriched by patient CSF antibodies. Coverage is divided into five amino acid bins and aligned to the full-length protein (NP_057663.1). The blue region represents the extracellular domain (ECD), the red regions represent LDLR-A domains within the ECD, the black region represents the transmembrane (TM) domain, and the orange region represents the intracellular domain (ICD). (B) Immunore-activity of patient CSF IgG (green) to HEK293T cells overexpressing a FLAG-tagged CD320 construct (red). Staining dilution = 1:25. Scale bar, 20 μm. (C) Control and CD320 KO primary human brain endothelial cells stained with a commercial CD320 antibody (left column) or patient CSF (middle and right columns) and DAPI (blue) to label nuclei. Scale bar, 20 μm. (D) Holotranscobalamin uptake in cells treated with case 1 CSF (red) or a commercial CD320 antibody (blue) compared with cells treated with healthy control CSF (black) (n = 2, paired one-way ANOVA with Tukey’s multiplicity correction, means ± SE). (E) Holotranscobalamin uptake in cells treated with control CSF (black) or case 1 CSF-enriched (magenta) or CSF-depleted (green) of anti-CD320 by affinity purification (n = 3, one-way ANOVA with Tukey’s multiplicity correction, means ± SE). (F) Vitamin B12 concentration in serum (red) and CSF (blue) of three healthy controls and case 1. (G) Representative images of brain endothelial cells treated with control or patient CSF, followed by staining for CD320 (red), wheat germ agglutinin (WGA, a plasma membrane marker; gray), and lysosomal-associated membrane protein (LAMP1; green). Arrows indicate yellow puncta where CD320 (red) colocalizes with lysosomes (LAMP1, green; scale bar = 20 μm). Quantification of CD320 colocalization with the LAMP1 (H) or WGA (I) in cells treated with control CSF (black), case 1 CSF (red), or a commercial anti-CD320 antibody (blue) (n = 3; one-way ANOVA; means ± SE).