1.

Cma ST8Sia IV polysialylates N- and O-glycoproteins. (A) Schematic representing the protein structure of Cma ST8Sia IV with localization and amino acid sequence of all 5 N-glycosylation sites predicted at positions Asn 50, 73, 118, 203, and 218. Positions of the transmembrane domain (TM) and sialylmotifs L, S, III, and VS are indicated. (B) Schematic representation of complex-type N-glycans of glycoproteins in HEK293 cells (upper) and oligomannose-type N-glycans of glycoproteins in MGAT1-deficient HEK293S cells (lower). Monosaccharides are represented according to the SNFG representation of glycans. (C) The Cma ST8Sia IV produced as a truncated form in the supernatant of transfected HEK293 and HEK293S cells were subjected to PNGase F treatment for various time points ranging from 0 to 30 min. An empty vector (Mock) was used as a negative control in both cell lines. After 5 min at 95 °C, proteins were separated on 8% SDS-PAGE and transferred onto a nitrocellulose membrane. WB was carried out with the anti-3 × FLAG antibody. Arrows on the right side represent the various N-glycosylation sites detected. The complete areas of the WB are shown in Supplementary Figure S3, including 5 additional time points (45, 60, 90, 120, and 240 min). (D) The initial polysialylation status of Cma ST8Sia IV produced in HEK293 or HEK293S cells was visualized by WB (left WB). Aliquots of the samples were treated with PNGase F to remove N-glycans. After 5 min at 65 °C, proteins were separated on 8% SDS-PAGE and transferred onto a nitrocellulose membrane. WB was carried out using the mAb735. The polysialylation status of glycoproteins after the sialylation reaction using 40 μM CMP-Neu5Ac was analyzed by WB in the same way (right WB). Molecular weight markers are indicated on the left side.