Mutations in STT3A and STT3B cause two congenital disorders of glycosylation

Abstract

We describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T > C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G > T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing.

Figure 1.

Identification of two consanguineous families with Sanger sequence confirmed mutations in STT3A and STT3B. (A) Family-1 pedigree shows two affected individuals from a consanguineous marriage where homozygosity mapping was performed on Case-1. Lower panel shows Sanger sequence confirmation of the STT3A c.1877T > C mutation (p.Val626Ala) in the affected male. Sample from the affected female was not available for testing. (B) Family-2 pedigree shows consanguinity and homozygosity mapping was performed on the affected male Case-2. Lower panel shows Sanger sequence confirmation of the STT3B c.1539 + 20G > T mutation. Filled in symbols denote affected individuals and filled in symbol with a line through it means individual is deceased.

Figure 2.

Reduced expression of STT3A and STT3B in Case-1 and Case-2 fibroblasts. (A) STT3A was immunoprecipitated from total cell extracts of fibroblasts (Case-1, CDG C-1; Case-2, CDG C-2; control, 42F and 50F) or HeLa cells that were pulse-labeled for 50 min with Tran-³⁵S label. Equal quantities of ³⁵S labeled protein were used for each immunoprecipitation. (B) Protein immunoblot detection of STT3B, ribophorin 1 (R 1), the ER chaperone BiP and the protein translocation channel (Sec61). Equal amounts of protein (100 µg) were electrophoresed in each lane. HeLa cell extracts serve as protein mobility controls. The asterisk designates a background band that migrates in the vicinity of STT3B. These results are representative of two individual experiments.

Figure 3.

STT3A and STT3B complementation measured by GFP fluorescence in cells. Cells from Case-1 (Fig. 3A) and Case-2 (Fig. 3B) were electroporated with the ER-GFP and the Glyc-ER-GFP in the presence of a control vector or vector expressing STT3A-DDK-His or STT3B-Myc-DDK. Anti-FLAG (DDK) antibody was used to identify transfected cells, and detected by a far-red linked secondary antibody. On top of its inherent green fluorescence, total GFP expression was monitored by a GFP antibody followed by a Cy3-linked secondary antibody. Images were monitored by fluorescent microscopy and processed using Image J software. The quantitation of the relative GFP fluorescence per cell is shown in the histogram and was calculated from at least 150 cells in each case. Error bars show standard deviation.

Figure 4.

Hypoglycosylation of STT3A-specific substrates in Case-1 fibroblasts. (A) A diagram of prosaposin showing the signal sequence (black), saposin domains (gray), glycosylation sites and disulfide bonds (black lines). (B) Control, CDG fibroblasts and HeLa cells were pulse labeled for 10 min with Tran-³⁵S label. The HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B for 72 h prior to pulse-labeling. EH designates digestion with endoglycosidase H. Prosaposin glycoforms (0–5 glycans) were immunoprecipitated with anti-saposin (D) sera and resolved by SDS–PAGE. (C) HeLa cells were co-transfected with STT3A siRNA and STT3A-DDK-His tagged expression vectors as indicated. STT3A and STT3A-DDK-His expression was evaluated by immunoprecipitation of pulse-labeled STT3A as in Figure 2A (upper panel). Prosaposin glycoforms (0–5 glycans) were immunoprecipitated with anti-saposin D sera and resolved by SDS–PAGE as in Figure 4B (lower panel). The quantitation for average number of glycans in each lane is determined by adding the intensities of each band and allocating a percentage contribution to each band. (D) A diagram of granulin showing the signal sequence (black), granulin domains (gray), glycosylation sites, disulfide bonds (black lines) and an alternatively spliced product (Gran2) that lacks granulin domains (C and D). (E) The long (pGran-1) and short (pGran-2) forms of the granulin precursor were immunoprecipitated from cells pulse labeled for 10 min with Tran-³⁵S label, using anti-granulin sera and resolved by SDS–PAGE. The pGran-1 glycoforms resolve poorly for unknown reasons.

Figure 5.

Hypoglycosylation of STT3B substrates in Case-2 fibroblasts. (A and C) Diagrams of sex hormone binding globulin (SHBG) (A) and β-GUSΔ123 (C) showing the N-terminal signal sequence, protease cleavage sites (arrowheads), free cysteine residues (black diamonds), disulfide bonds (black lines) and C-terminal glycosylation sites. (B and D) HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B as indicated for 48 h. Fibroblasts (control, Case-1 and Case-2) and siRNA treated HeLa cells were transfected with expression vectors for SHBG (B) or Myc-DDK tagged β-GUSΔ123 (D) 24 h before pulse-labeling for 10 min with Tran-³⁵S label. (B) SHBG glycoforms (0–2 glycans) were immunoprecipitated with anti-SHBG sera and resolved by SDS–PAGE. (D) β-GUSΔ123 glycoforms (0 or 1 glycan) were immunoprecipitated with anti-DDK sera and resolved by SDS–PAGE. EH designates digestion with endoglycosidase H.