Richard Steet

Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder.

The congenital disorders of glycosylation (CDG) are characterized by defects in N-linked glycan biosynthesis that result from mutations in genes encoding proteins directly involved in the glycosylation pathway. Here we describe two siblings with a fatal form of CDG caused by a mutation in the gene encoding COG-7, a subunit of the conserved oligomeric Golgi (COG) complex. The mutation impairs integrity of the COG complex and alters Golgi trafficking, resulting in disruption of multiple glycosylation pathways. These cases represent a new type of CDG in which the molecular defect lies in a protein that affects the trafficking and function of the glycosylation machinery.

The iminosugar isofagomine increases the activity of N370S mutant acid β-glucosidase in Gaucher fibroblasts by several mechanisms

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid β-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 ± 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Strain‐Promoted Alkyne–Azide Cycloadditions (SPAAC) Reveal New Features of Glycoconjugate Biosynthesis

We have shown that 4‐dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a CuI catalyst with azido‐containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide–alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal‐free alkyne–azide fluoro‐switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido‐containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O‐glycan sialylation than galactosylation, whereas sialylation and galactosylation of N‐glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins.

Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells

Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represent a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location, and in particular it has been shown that DIBO derivative 4 can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.

A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

PMM2-CDG patients have phosphomannomutase (Pmm2) deficiency, with developmental and N-linked glycosylation defects attributed to depletion of mannose-1-phosphate and downstream lipid-linked oligosaccharides (LLOs). This, the first PMM2-CDG zebrafish model, shows, unexpectedly, that accumulation of the Pmm2 substrate mannose-6-phosphate explains LLO deficiency.

A splicing mutation in the α/β GlcNAc-1-phosphotransferase gene results in an adult onset form of mucolipidosis III associated with sensory neuropathy and cardiomyopathy†

A 47‐year‐old female who presented with a dilated cardiomyopathy and mild neuropathy was found to have pseudoHurler polydystrophy (mucolipidosis III). The serum lysosomal enzymes were strikingly elevated and GlcNAc‐1‐phosphotransferase activity in the patients fibroblasts was 3% of normal. Sequence analysis of the patients genomic DNA revealed a homozygous mutation of the last nucleotide of the 135‐bp exon 7 of the phosphotransferase gene encoding the α/β subunits, resulting in aberrant splicing and skipping of this exon. Remarkably, none of the skeletal and connective tissue anomalies characteristic of the disease were present. This case is the first example of mucolipidosis III presenting in an adult patient and further broadens the clinical spectrum of the disease.

Selective Exo‐Enzymatic Labeling of N‐Glycans on the Surface of Living Cells by Recombinant ST6Gal I

A game of tag: N-Glycans on the surface of living cells were selectively tagged by exogenously administering recombinant ST6Gal I sialyltransferase and azide-modified CMP-Neu5Ac. This modification was followed by a strain-promoted cycloaddition using a biotin-modified dibenzylcyclooctynol (red star=biotin). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease.

Altered chondrocyte differentiation and extracellular matrix homeostasis in a zebrafish model for mucolipidosis II.

Mucolipidosis II (ML-II) is a pediatric disorder caused by defects in the biosynthesis of mannose 6-phosphate, the carbohydrate recognition signal responsible for targeting certain acid hydrolases to lysosomes. The mechanisms underlying the developmental defects of ML-II are largely unknown due in part to the lack of suitable animal models. To overcome these limitations, we developed a model for ML-II in zebrafish by inhibiting the expression of N-acetylglucosamine-1-phosphotransferase, the enzyme that initiates mannose 6-phosphate biosynthesis. Morphant embryos manifest craniofacial defects, impaired motility, and abnormal otolith and pectoral fin development. Decreased mannose phosphorylation of several lysosomal glycosidases was observed in morphant lysates, consistent with the reduction in phosphotransferase activity. Investigation of the craniofacial defects in the morphants uncovered striking changes in the timing and localization of both type II collagen and Sox9 expression, suggestive of an accelerated chondrocyte differentiation program. Accumulation of type II collagen was also noted within misshapen cartilage elements at later stages of development. Furthermore, we observed abnormal matrix formation and calcium deposition in morphant otoliths. Collectively, these data provide new insight into the developmental pathology of ML-II and suggest that altered production and/or homeostasis of extracellular matrix proteins are integral to the disease process. These findings highlight the potential of the zebrafish system in studying lysosomal disease pathogenesis.

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Niemann–Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.

Analysis of Mucolipidosis II/III GNPTAB Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme GlcNAc-1-phosphotransferase Involved in Catalytic Function and Lysosomal Enzyme Recognition

Background: Mutations in GNPTAB cause the lysosomal disorders mucolipidosis II and III αβ. Results: All reported missense mutations were studied and showed various consequences on its gene product, αβ GlcNAc-1-phosphotransferase. Conclusion: Domains responsible for catalytic activity and lysosomal hydrolase recognition were identified. Significance: Analysis of patient mutations provided new insight into the functional domains of αβ GlcNAc-1-phosphotransferase. UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.