Jos Jansen

42 Chapter 2 incomplete glycans lacking sialic acid (79,265 amu, peak 2), galactose (79,104 amu, peak 3), and additional minor isoforms lacking sialic acid and/or galactose in individual F1-II1 (Figure 2C, peaks 4–8, see Table S7 for a list of all annotated glycan structures). This pattern is compatible with an overall detrimental effect on Golgi glycosylation. Together with abnormal O-glycosylation, these data are suggestive of a Golgi homeostasis defect and a combined disorder of N-glycosylation and mucin-type O-glycosylation. Metabolic Labeling of Sialic Acids We further assessed glycosylation efficiency in skin-derived fibroblasts by metabolic labeling of sialic acids with alkyne tagged synthetic sugar analogs. These alkyne-tagged sialic acids are incorporated into nascent glycoproteins by the cell allowing for specific detection with fluorescently labeled azides.(23) In CCDC115-deficient individuals F1-II1 and F1-II2, the pool of glycoconjugates was located to the Golgi, and immunofluorescence signal quantification in the Golgi showed a clear reduction for all individuals, in agreement with less efficient Golgi glycosylation (Figures 3A and 3B). Additionally, we observed a dispersed pattern of the glycoconjugates, suggestive of dilatation of the Golgi (Figure 3B). ► Figure 2. Glycosylation abnormalities in CCDC115 deficient patients. (A) The isofocusing profiles (IEF) of serum transferrin (Tf) and serum apolipoprotein CIII (aCIII). Tf has two N-glycosylation sites and the most abundant fraction corresponds with four sialic acids. aCIII has one O-linked glycan that can host one or two sialic acids. The accompanying numbers represent the total number of sialic acids in the different protein isoforms. All patients show a relative increase of isoforms Tf-2 and Tf-3 and of aCIII-1. For patient 2.1 HPLC was used to assess Tf glycosylation status. (B) MALDI mass spectrometry profiles of total serum N-glycans of a healthy control and CCDC115 deficient patient 1.1. An increase in hypoglycosylated glycans can be seen for all patients when compared to the control. Most notably the glycans with theoretical masses of 2433 m/z and 2228 m/z are increased, indicating loss of one sialic acid (2433) or one sialic acid plus one galactose (2228). (C) The nanochip-C8-QTOF mass profile is shown of the intact transferrin protein with two attached glycans at 79555 amu (peak 1). Any subsequent sialic acid and/or hexose loss can be calculated based on mass difference with the main peak (i.e., loss of one sialic acid (purple diamond, peak 2). Patient 1.1 shows a reduction in sialic acid and galactose residues (peaks 3 to 8). (D) Incubation of healthy control fibroblasts with the alkynyl-tagged sialic acid precursor ManNAl which can be labeled with azide-conjugated fluorophores. A reduced fluorescent signal can be visualized for patient 1.1 and 1.2. Scale bars indicate 75 µm. Percentage reduction in metabolic labeling for the analyzed patients compared to control cells (approximately 100 cells were counted per condition).