Human iPSC-derived astrocytes generated from donors with globoid cell leukodystrophy display phenotypes associated with disease.

Globoid cell leukodystrophy (Krabbe disease) is a fatal neurodegenerative, demyelinating disease caused by dysfunctional activity of galactosylceramidase (GALC), leading to the accumulation of glycosphingolipids including psychosine. While oligodendrocytes have been extensively studied due to their high levels of GALC, the contribution of astrocytes to disease pathogenesis remains to be fully elucidated. In the current study, we generated induced pluripotent stem cells (iPSCs) from two donors with infantile onset Krabbe disease and differentiated them into cultures of astrocytes. Krabbe astrocytes recapitulated many key findings observed in humans and rodent models of the disease, including the accumulation of psychosine and elevated expression of the pro-inflammatory cytokine IL-6. Unexpectedly, Krabbe astrocytes had higher levels of glucosylceramide and ceramide, and displayed compensatory changes in genes encoding glycosphingolipid biosynthetic enzymes, suggesting a shunting away from the galactosylceramide and psychosine pathway. In co-culture, Krabbe astrocytes negatively impacted the survival of iPSC-derived human neurons while enhancing survival of iPSC-derived human microglia. Substrate reduction approaches targeting either glucosylceramide synthase or serine palmitoyltransferase to reduce the sphingolipids elevated in Krabbe astrocytes failed to rescue their detrimental impact on neuron survival. Our results suggest that astrocytes may contribute to the progression of Krabbe disease and warrant further exploration into their role as therapeutic targets.

Mapping Proteoglycan Function Using Novel Genetic Strategies.

Multiple intrinsic and extrinsic factors contribute to stem and neuronal precursor cell maintenance and/or differentiation. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. The complexity and diversity of the glycan structure of proteoglycans make their functional characterization a challenging task. In order to test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation, we have taken a loss-of-function approach by developing a library of both biosynthetic and degradative enzymes to specifically remodel the ECM.

Computational and experimental analysis of the glycophosphatidylinositol-anchored proteome of the human parasitic nematode Brugia malayi.

Further characterization of essential systems in the parasitic filarial nematode Brugia malayi is needed to better understand its biology, its interaction with its hosts, and to identify critical components that can be exploited to develop novel treatments. The production of glycophosphatidylinositol-anchored proteins (GPI-APs) is essential for eukaryotic cellular and physiological function. In addition, GPI-APs perform many important roles for cells. In this study, we characterized the B. malayi GPI-anchored proteome using both computational and experimental approaches. We used bioinformatic strategies to show the presence or absence of B. malayi GPI-AP biosynthetic pathway genes and to compile a putative B. malayi GPI-AP proteome using available prediction programs. We verified these in silico analyses using proteomics to identify GPI-AP candidates prepared from the surface of intact worms and from membrane enriched extracts. Our study represents the first description of the GPI-anchored proteome in B. malayi and lays the groundwork for further exploration of this essential protein modification as a target for novel anthelmintic therapeutic strategies.

Mapping proteoglycan functions with glycosidases.

The intrinsic and extrinsic factors that contribute to stem and neuronal precursor cell maintenance and/or differentiation remain poorly understood. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. We have taken a loss-of-function approach, by developing a library of bacterial lyases and sulfatases to specifically remodel the ECM and test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation.

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.

Complementation of essential yeast GPI mannosyltransferase mutations suggests a novel specificity for certain Trypanosoma and Plasmodium PigB proteins.

The glycosylphosphatidylinositol (GPI) anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH2-CH2-CH2-PO4-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-myo-inositol-PO4-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2)-linked fourth mannose (Man-IV) that is side-branched to the third mannose (Man-III) of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2)-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man4-GPIs) being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the in vivo specificity of Gpi10/PigB proteins from several Plasmodium and Trypanosoma species by examining their ability to restore viability to Saccharomyces cerevisiae strains harboring lethal defects in Man-III (gpi10Δ) or Man-IV (smp3Δ) addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from T. cruzi, T. congolense and P. falciparum are each capable of separately complementing essential gpi10Δ and smp3Δ mutations, while PIGB genes from T. vivax and T. brucei only complement gpi10Δ. Additionally, we show the ability of T. cruzi PIGB to robustly complement a gpi10Δ/smp3Δ double mutant. Our data suggest that certain Plasmodium and Trypanosoma PigB mannosyltransferases can transfer more than one mannose to GPI precursors in vivo, and suggest a novel biosynthetic mechanism by which Man4-GPIs may be synthesized in these organisms.

In vivo incorporation of an azide-labeled sugar analog to detect mammalian glycosylphosphatidylinositol molecules isolated from the cell surface.

N-Acetylgalactosamine (GalNAc) linked to the first mannose of glycosylphosphatidylinositol (GPI) core has been previously reported to be heterogeneously present on some mammalian GPI-anchored proteins. Here we present a method for profiling GalNAc-containing GPI-anchored proteins in mammalian cells by metabolic labeling with tetraacetylated N-azidoacetylgalactosamine (GalNAz) followed by biotinylation of the incorporated sugar analog. We have labeled both endogenous and recombinant GPI-anchored proteins with GalNAz, and demonstrated that the azide-activated sugar gets incorporated into the GPI glycan, likely as an unsubstituted side branch of the core structure. GalNAz was detected only on GPI molecules attached to proteins, and not on GPI precursors, indicating that GalNAc modification takes place after the GPI anchor is transferred to protein. We have highlighted the utility of this cell labeling approach by demonstrating the ability to examine specific GalNAc-containing GPI-anchored proteins isolated non-destructively from separate membrane domains (apical and basolateral) in polarized epithelial cells. This study represents the first demonstration of site-specific in vivo labeling of a GPI moiety with a synthetic sugar analog.