A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation - PubMed (original) (raw)

. 2014 Jan 15;23(2):418-33.

doi: 10.1093/hmg/ddt434. Epub 2013 Sep 10.

Kazuhiro Aoki, Heather Flanagan-Steet, Chin-Fu Chen, Xiang Fan, Frank Bartel, Marharyta Petukh, Ayla Pittman, Robert Saul, Alka Chaubey, Emil Alexov, Michael Tiemeyer, Richard Steet, Charles E Schwartz

Affiliations

A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation

Luigi Boccuto et al. Hum Mol Genet. 2014.

Abstract

'Salt & Pepper' syndrome is an autosomal recessive condition characterized by severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial features and altered dermal pigmentation. High-density SNP array analysis performed on siblings first described with this syndrome detected four shared regions of loss of heterozygosity (LOH). Whole-exome sequencing narrowed the candidate region to chromosome 2p11.2. Sanger sequencing confirmed a homozygous c.994G>A transition (p.E332K) in the ST3GAL5 gene, which encodes for a sialyltransferase also known as GM3 synthase. A different homozygous mutation of this gene has been previously associated with infantile-onset epilepsy syndromes in two other cohorts. The ST3GAL5 enzyme synthesizes ganglioside GM3, a glycosophingolipid enriched in neural tissue, by adding sialic acid to lactosylceramide. Unlike disorders of glycosphingolipid (GSL) degradation, very little is known regarding the molecular and pathophysiologic consequences of altered GSL biosynthesis. Glycolipid analysis confirmed a complete lack of GM3 ganglioside in patient fibroblasts, while microarray analysis of glycosyltransferase mRNAs detected modestly increased expression of ST3GAL5 and greater changes in transcripts encoding enzymes that lie downstream of ST3GAL5 and in other GSL biosynthetic pathways. Comprehensive glycomic analysis of N-linked, O-linked and GSL glycans revealed collateral alterations in response to loss of complex gangliosides in patient fibroblasts and in zebrafish embryos injected with antisense morpholinos that targeted zebrafish st3gal5 expression. Morphant zebrafish embryos also exhibited increased apoptotic cell death in multiple brain regions, emphasizing the importance of GSL expression in normal neural development and function.

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Figures

Figure 1.

Figure 1.

Glycosphingolipid biosynthetic pathways. Mammalian GSL biosynthesis begins with the glycosylation of ceramide that is either galactosylated to form GalCer or glucosylated to form GlcCer. GlcCer is subsequently elongated by the addition of Gal to form LacCer, the substrate for ST3GAL5. Sialylation of LacCer by this enzyme, which is deficient in S&P syndrome, produces the ganglioside GM3, the precursor for formation of all complex gangliosides of the a and b type (boxed in red). S&P syndrome patients lack all of these gangliosides, but are still able to produce the c type gangliosides as well as globo-, lacto- and neolactoseries GSLs. Glycan symbolic representations are consistent with the recommendations of the Glycobiology community as described by Varki, et al. (

www.ncbi.nlm.nih.gov/books/NBK1931/figure/ch1.f5/?report=objectonly

). CGT, ceramide glucosyltransferase; B4GALT6, β4-galactosyltransferase 6; B4GALNT1, β4-_N_-acetylgalactaminyltransferase 1; B3GALT4, β3-galactosyltransferase 4; ST3GAL2, α3-sialyltransferase 2; ST3GAL5, α3-sialyltransferase 5 (GM3 synthase); A4-GALT, α4-galactosyltransferase; B3GNT5, β3-_N_-acetylglucosaminyltransferase 5.

Figure 2.

Figure 2.

Pedigree and clinical presentation of Family K6848 with Salt and Pepper syndrome. (A) Partial pedigree of Family K6848. The proband, III-5, is indicated by the arrow. Photographs of the three siblings affected with S&P syndrome are shown below the pedigree (ID denotes intellectual deficiency; CP denotes cerebral palsy). (B) Closer view of the hands of Patient III-4 showing pigmentary findings and (C) the feet of III-5 showing hypertonicity.

Figure 3.

Figure 3.

The ST3GAL5 mutation in S&P syndrome generates a p.E322K missense mutation within a highly conserved sialyltransferase domain. (A) ST3GAL5, like all other known mammalian sialyltransferases, is a type 2 transmembrane protein with a very small cytosolic domain (TM denotes transmembrane domain). The portion of the protein found within the Golgi lumen possesses a stalk domain that extends the carboxy-terminal, globular catalytic domain away from the Golgi membrane. All sialyltransferases possess four signature domains: the L-motif (long motif, green), the S-motif (short motif, pink), motif 3 (blue) and the VS-motif (very short motif, turquoise). (B) A survey of known ST3GAL5 S-motif sequences is aligned. Numbers indicate the starting residue of the presented sequences. In S&P syndrome, a c.994G>A transition results in an E>K missense mutation (highlighted in red) at a highly conserved residue within the sialyltransferase S-motif. The amino acids defining the conserved S-motif are boxed in pink and amino acid residues conserved across all identified ST3GAL5 proteins are indicated by a dot. The asterisk in the diagram indicates the approximate position of a mutation previously identified in Old Amish and French cohorts that cause a premature translational stop (see Discussion for more details). The accession numbers for the presented ST3GAL5 sequences are as follows: Homo sapiens NP_001035902.1; Pan troglodytes NP_001032378.1; Macaca mulatta NP_001244429.1; Mus musculus NP_035505.2; Gallus gallus NP_001001192.1; Takifugu rubripes XP_003978941.1; Danio rerio AAI64193.1; Felis catus XP_003984278.1. (C and D) The sequences of the wild-type (C) and S&P form (D) of ST3GAL5 were threaded onto the crystal structure of porcine ST3GAL1. The p.E332K mutation disrupts interactions between E332 and three basic residues (K95, R98 and R171). The replacement of the anionic carboxylate of the E residue with the amine of the K residue requires the S&P form of ST3GAL5 to accommodate an additional positive charge within a cluster of basic residues (K95, R98, R171), and, as a result, is predicted to partially unfold the protein.

Figure 4.

Figure 4.

TLC analysis of GSLs extracted from control and S&P patient III-5 fibroblasts. Glycosphingolipid representations on the left indicate the migration positions of standards shown in the Neutral and Ganglioside lanes. The asterisk indicates GM3, which was detected in Control but not in S&P patient III-5 fibroblasts. In the absence of complex ganglioside biosynthesis, the GSL profile of III-5 fibroblast is enriched in globo-series products (Gb3- and Gb4Cer).

Figure 5.

Figure 5.

Mass spectrometric analysis of GSLs extracted from control and S&P patient III-5 fibroblasts. (A) Full mass spectrum (MS) of permethylated GSLs extracted from control fibroblasts shows predominance of the neutral Gb3Cer and the ganglioside GM3 (asterisk). The TLC lanes shown in Figure 4 are reproduced turned on their side for reference. Glycosphingolipids are detected as families of structures distinguished by their lipid compositons (red brackets). In some cases, these families overlap with each other (GM3 and Gb4Cer, for example), but individual peaks can be assigned to a specific lipid family based on MS/MS and MSn characterization of the associated glycan structure. The arrow indicates LacCer, the precursor for GM3. (B) Full MS of permethylated GSLs extracted from S&P III-5 fibroblasts shows a complete loss of GM3 and increase in the abundance of Gb4, while the relative abundance of LacCer is unchanged compared with control. The lipid distribution for each of the S&P GSL families is also shifted toward greater abundance of longer chain lipids.

Figure 6.

Figure 6.

The glycoprotein O-linked glycan profile is enriched for highly sialylated structures in S&P III-5 fibroblasts. After β-elimination and permethylation, the relative abundance of the indicated glycans was measured by MS. In the S&P fibroblasts, the amount of the core 1 disaccharide and its monosialylated derivative (glycans numbered 1 and 2, respectively) are decreased relative to control, while the disialylated form of the same structure (glycan number 6) is increased, indicating potentially compensatory shifts in glycoprotein sialylation as glycolipid sialylation decreases. A similar shift toward greater sialylation is seen on core 2 structures as well (glycans numbered 4, 7, 8 and 9).

Figure 7.

Figure 7.

The glycoprotein N-linked glycan profile is enriched for sialylated, complex glycans in S&P III-5 fibroblasts. After release by PNGaseF and permethylation, the relative abundance of the indicated glycans was measured by MS. Complex, sialylated glycans (highlighted in purple) are increased and less processed high mannose structures (highlighted in green) are decreased in comparison with control.

Figure 8.

Figure 8.

Knockdown of st3gal5, but not overexpression, results in increased neuronal cell death in zebrafish embryos. Zebrafish embryos were injected with either antisense morpholinos against st3gal5, st3gal5 mRNA, or both and were subsequently analyzed for gross morphology and stained with acridine orange to assess cell death in the brain. Gross morphology of 3 dpf embryos was assessed by light microscopy (left column). Acridine orange staining of 3 dpf embryos was assessed by fluorescence microscopy at 50× magnification (middle column) and at 200× magnification (right column) of the head region. Compared with wild-type (WT), acridine orange staining reveals increased cell death in morpholino-injected embryos (ST3GAL5 MO). Cell death was rescued by co-injection of untargeted ST3GAL5 mRNA (ST3GAL5 MO + RNA). Gain-of-function phenotypes were not detected with increased enzyme expression, achieved by injection of st3gal5 mRNA into wild-type embryos (mRNA alone).

Figure 9.

Figure 9.

Knockdown of st3gal5 decreases GSL complexity in zebrafish embryos. (A) The full MS GSL profile of wild-type (WT) embryos 3 days post fertilization (dpf) is dominated by hexaosylceramide and GM3. (B) Morpholino knockdown of st3gal5 (ST3GAL5 MO) decreases GM3 levels at 3 dpf and increases LacCer, the precursor for GM3. (C) At 5 dpf, wild-type GSL profiles become diversified by the appearance of complex, highly sialylated species. (D) st3gal5 MO embryos at 5 dpf are greatly reduced in GM3 and in all of the more complex sialylated GSLs, while LacCer remains elevated in st3gal5 MO embryos at 5 dpf.

Figure 10.

Figure 10.

Glycosphingolipid levels respond to st3gal5 dosage in zebrafish embryos. The relative abundances of 5 GSLs that lie along the two branching biosynthetic pathways are shown as detected in 3 dpf zebrafish embryos. LacCer is the precursor for the formation of GA2 and GM3. Knockdown of st3gal5 (MO) reduces GM3 levels, which is expected to decrease the flux of LacCer entering into the formation of GM3, GD3 GT3, and more complex gangliosides. Accordingly, LacCer and GA2 levels are increased in the morphant. The increase in LacCer and the decreases in GM3 as well as the more highly sialylated gangliosides (GD3, GT3) can be titrated toward normal levels by expressing WT st3gal5 in the morpholino background (MO + wtRNA). The production of GM3, and more highly sialylated gangliosides, is further enhanced by expression of wild-type st3gal5 in the WT background (wtRNA). Injection of the S&P mutant RNA (mutRNA) into WT embryos has no effect on GSL production, indicating that the S&P enzyme lacks a dominant negative effect on biosynthesis.

Figure 11.

Figure 11.

The glycoprotein O-linked glycan profile is enriched for highly sialylated structures in zebrafish st3gal5 morphant embryos. (A) O-linked glycans were released by β-elimination from glycoproteins harvested from 3 dpf embryos that were either WT or ST3GAL5 morphants (MO). Following permethylation, glycan profiles were characterized by mass spectrometry. Core 1 disaccharide (glycan number 1), as well as the monosialylated forms of the core 1 disaccharide (glycans numbered 2) were decreased in the morphant, while disialylated and multisialylated core 1 was increased (glycans numbered 5, 23 and 25). (B) At 5 dpf, the shift toward greater O-linked glycan sialylation was attenuated.

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