Deficiency in ST6GAL1, one of the two α2,6-sialyltransferases, has only a minor effect on the pathogenesis of prion disease

Abstract

Prion diseases are a group of fatal neurodegenerative diseases caused by misfolding of the normal cellular form of the prion protein or PrP^C, into a disease-associated self-replicating state or PrP^Sc. PrP^C and PrP^Sc are posttranslationally modified with N-linked glycans, in which the terminal positions occupied by sialic acids residues are attached to galactose predominantly via α2-6 linkages. The sialylation status of PrP^Sc is an important determinant of prion disease pathogenesis, as it dictates the rate of prion replication and controls the fate of prions in an organism. The current study tests whether a knockout of ST6Gal1, one of the two mammalian sialyltransferases that catalyze the sialylation of glycans via α2-6 linkages, reduces the sialylation status of PrP^Sc and alters prion disease pathogenesis. We found that a global knockout of ST6Gal1 in mice significantly reduces the α2-6 sialylation of the brain parenchyma, as determined by staining with Sambucus Nigra agglutinin. However, the sialylation of PrP^Sc remained stable and the incubation time to disease increased only modestly in ST6Gal1 knockout mice (ST6Gal1-KO). A lack of significant changes in the PrP^Sc sialylation status and prion pathogenesis is attributed to the redundancy in sialylation and, in particular, the plausible involvement of a second member of the sialyltransferase family that sialylate via α2-6 linkages, ST6Gal2.

Keywords: N-glycosylation; ST6GAL1; ST6GAL2; mouse; prion; prion diseases; sialic acid; sialyltransferases.

FIGURE 1

The global knockout of ST6Gal1. (A) Western blot of 2% spleen tissues from ST6Gal1-KO (KO) and control C57Bl/6 (WT) mice stained with anti-ST6Gal1 antibody (#AF5924, R&D Systems, Minneapolis, MN). (B) RT-qPCR analysis of ST6Gal1 expression using Bio-Rad primers (qMmuCID0009827) or primers to Exon 2 of the gene in cortex (Ctx), hippocampus (Hp), thalamus (Th) and midbrain (MB) from ST6Gal1-KO (KO) and control C57Bl/6 (WT) mice. Each sample was analyzed in triplicate. The mean and standard deviation are shown. TBP was used as a housekeeping gene. (C) Staining of 22L-infected brains of ST6Gal1-KO (KO) and C57Bl/6 (WT) mice, euthanized at 146 and 145 days post-inoculation (dpi), respectively, using Sambucus Nigra lectin (SNA). Scale bar = 1 mm.

FIGURE 2

A modest impact of ST6Gal1 knockout on the incubation time to disease. (A) Kaplan-Meier survival plot for ST6Gal1-KO and C57Bl/6 (WT) mice inoculated IC with 1% scrapie brain homogenate of the 22L strain. p value was calculated using Mantel-Cox test for comparison of survival curves. (B) Western blot analysis and quantification of PrP^Sc in brain homogenates (BH) and spleen homogenates (SH) of ST6Gal1-KO and C57Bl/6 (WT) mice inoculated with 22L. Samples were treated with PK. Untreated (-PK) samples were used as a reference, and were loaded as a 10-fold dilution. Western blots were stained with the ab3531 antibody. PrP^Sc amount in brains and spleens were compared using unpaired t-tests and presented as individual animals and means (n = 10 WT, n = 7 ST6Gal1-KO, *p < 0.05). (C) The percentage of di-, mono- and unglycosylated brain-derived PrP^Sc from ST6Gal1-KO and C57Bl/6 (WT) mice inoculated with 22L. Data presented as individual animals and means (n = 10 WT and n = 7 ST6Gal1-KO).

FIGURE 3

Histopathological analysis of 22L-infected mouse brains. Vacuolation revealed with hematoxylin-eosin (H&E) staining, deposition of PrP^Sc stained with SAF-84 antibody, and reactive microglia stained for Iba1 in ST6Gal1-KO and C57Bl/6 (WT) mice inoculated IC with 22L and euthanized at 146 and 145 dpi, respectively. Staining of normal C57Bl/6 (WT) euthanized at 212 days of age is shown as references (right panels).

FIGURE 4

Analysis of the sialylation status of PrP^Sc. Representative 2D Western blot of brain-derived PrP^Sc from ST6Gal1-KO and C57Bl/6 (WT) mice euthanized at 146 and 145 dpi, respectively (A), and spleen-derived PrP^Sc from ST6Gal1-KO and C57Bl/6 (WT) mice euthanized at 158 and 154 dpi, respectively (B). Mice were inoculated IC with 1% scrapie brain homogenate of the 22L strain. Middle panels show brain- and spleen-derived PrP^Sc from WT mice desialylated using sialidase Arthrobacter ureafaciens. Prior to 2D blot, brain and spleen materials were treated with 20 μg/mL PK. Anti-PrP antibody ab3531 was used for immunodetection. Arrows point at di-, mono- and non-glycosylated glycoforms.