RPTPζ/phosphacan is abnormally glycosylated in a model of muscle–eye–brain disease lacking functional POMGnT1
Highlights
► A novel hypoglycosylated protein in POMGnT1 knockout brains, a model of CMDs. ► RPTPζ/phosphacan is a likely substrate for O-mannosylation by POMGnT1 in vivo. ► HNK-1 glycans on RPTPζ/phosphacan are diminished in POMGnT1 knockouts. ► Glycosylation of RPTPζ/phosphacan is unaffected in LARGE knockouts.
Introduction
Congenital muscular dystrophies (CMDs) with associated brain abnormalities are a group of devastating autosomal recessive disorders characterized by congenital muscular dystrophy, type II lissencephaly and eye anomalies (Dobyns et al., 1985, Haltia et al., 1997, Jimenez-Mallebrera et al., 2005, Reed, 2009). Abnormalities in protein O-mannosyl glycosylation, often arising from mutations in genes encoding functional or putative glycotransferase involved in O-mannosyl glycosylation, are causal to this group of disorders (Brockington et al., 2001a, Brockington et al., 2001b, Beltran-Valero de Bernabe et al., 2002, Schessl et al., 2006). Exemplary to this is muscle–eye–brain disease (MEB) which arises from mutations in the gene encoding the known glycotransferase protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) (Yoshida et al., 2001, Liu et al., 2006). POMGnT1 catalyzes the transfer of an N-acetylglucosamine saccharide on to O-mannose present on serine and threonine residues of glycoproteins (Yoshida et al., 2001).
The most well characterized protein substrate of O-mannosyl glycosylation is α-dystroglycan (α-DG), which forms a heterodimer with β-dystroglycan to function as a transmembrane cell-surface receptor for constituents of the extracellular matrix (ECM) present in the basal lamina of the developing and mature brain (Ervasti and Campbell, 1991, Ervasti and Campbell, 1993, Ibraghimov-Beskrovnaya et al., 1992, Montanaro et al., 1999). Proper protein O-mannosyl glycosylation of α-DG is essential to maintain its ligand-binding properties and the subsequent integrity of the basement membrane (Kano et al., 2002, Michele et al., 2002, Kim et al., 2004). Disruption of pial basement membrane integrity consequently leads to overmigration of cortical neurons and type II lissencephaly along with other CNS pathologies (Michele et al., 2002). A number of studies have demonstrated that altered glycosylation of α-DG accounts for most of the obvious neural abnormalities (Moore et al., 2002, Satz et al., 2010), however a number of pieces of evidence indicate that additional protein substrates for protein O-mannosyl glycosylation exist and may also contribute to the phenotypes observed in these disorders. Glycomic analysis revealed brains of animals lacking α-DG have similar amounts of proteins with O-mannose-initiated structures as wild-type animals (Stalnaker et al., 2011). Work also showed that up to 30% of all O-linked sugars in the brain are O-linked via mannose (Finne et al., 1979, Krusius et al., 1986, Chai et al., 1999, Kogelberg et al., 2001), a number that could not be accounted for by α-DG alone. Furthermore, O-mannosyl-linked carbohydrates have also been described on particular cell-adhesion molecules (Bleckmann et al., 2009). Therefore identifying and characterizing novel protein substrates with altered glycosylation in these CMDs may provide important insights into the molecular basis of their complex phenotypes. Perhaps most importantly, future avenues of therapeutic intervention may be revealed in the yet-unidentified molecular underpinnings of these disorders.
Our previous work demonstrated that the monoclonal antibody, Cat-315, likely detects a Human Natural Killer-1 (HNK-1) epitope present on receptor protein tyrosine phosphatase ζ (RPTPζ), also referred to as RPTPβ, in the developing brain with biochemical properties that correlated with O-linked mannose structures (Matthews et al., 2002, Dino et al., 2006) and in a neuroblastoma cell line (Abbott et al., 2008). RPTPζ along with its secreted variant, phosphacan (Maurel et al., 1994), are primarily expressed in the CNS (Shitara et al., 1994, Maeda et al., 1995) and have been implicated in several key developmental neural processes including proliferation (Ida et al., 2006, Soh et al., 2007), differentiation (Canoll et al., 1996, Ranjan and Hudson, 1996, Meng et al., 2000, Soh et al., 2007) cell-adhesion and migration (Abbott et al., 2008), axonal guidance and neurite outgrowth (Grumet et al., 1996, Sakurai et al., 1997, Hayashi et al., 2005), myelination (Harroch et al., 2000, Harroch et al., 2002) and higher order cognitive function (Niisato et al., 2005). The extracellular domains of RPTPζ/phosphacan bind a wide array of ligands important for normal CNS development (for review see (Peles et al., 1998)) including pleiotrophin (Maeda et al., 1996, Meng et al., 2000), midkine (Maeda et al., 1999), tenascin (Grumet et al., 1994, Milev et al., 1995), NCAM, Ng-CAM (Maurel et al., 1994, Milev et al., 1994) and contactin (Sakurai et al., 1997). Previous work has demonstrated that carbohydrate modifications on RPTPζ modulate receptor–ligand binding which can in-turn influence downstream signaling and cellular behaviors (Milev et al., 1995, Peles et al., 1998, Maeda et al., 2003, Abbott et al., 2008). Therefore, altered O-mannosylation of RPTPζ/phosphacan could potentially have deleterious effects on neural development.
The work presented herein demonstrates that RPTPζ/phosphacan is hypoglycosylated in POMGnT1 knockout mice, an animal model of MEB. These data suggest that RPTPζ/phosphacan is a novel and major protein substrate for protein O-mannosyl glycosylation by POMGnT1 in vivo. Furthermore we demonstrate that in the developing brain RPTPζ/phosphacan bears the majority of HNK-1 reactivity in the soluble fraction, and most of these glycans on RPTPζ/phosphacan are O-linked via mannose. Given the CNS-enriched expression and interactions of RPTPζ/phosphacan and the known importance of the HNK-1 carbohydrate in development, our data argue for an important role of altered O-mannosyl glycosylation on RPTPζ/phosphacan and a potential contribution to the complex neurological phenotypes associated with CMDs.
Section snippets
Animals
Protocols for animal usage were approved by the Institutional Animal Care and Use Committee of Upstate Medical University. POMGnT1 knockout mice were generated by Lexicon Genetics Incorporated (The Woodlands, TX, USA) (Liu et al., 2006). Large myodystrophy (Largemyd) mutant mice were acquired from Jackson Laboratories (Bar Harbor, ME, USA). For timed pregnant breeding, the morning of plug discovery was designated E0. POMGnT1 and Largemyd genotyping was carried out as previously published (
The immunoreactivity of monoclonal antibodies Cat-315 and 6B4 is dramatically reduced in the cortex of embryonic and early postnatal POMGnT1 knockout animals
Previous work definitively demonstrated the monoclonal antibody Cat-315 prominently detects an epitope on aggrecan in the adult brain (Matthews et al., 2002), but an epitope on RPTPζ/phosphacan in the immature brain (Dino et al., 2006). Further characterization showed that the Cat-315 antibody detects an O-linked carbohydrate epitope (Matthews et al., 2002, Dino et al., 2006), which we hypothesized was likely O-linked via mannose. To test this hypothesis, Cat-315 reactivity was evaluated in
Abnormal glycosylation of RPTPζ/phosphacan in models of CMDs with associated brain abnormalities
The congenital nature of CMD disorders, which stem from abnormalities in prenatal development provide a significant challenge to both diagnosing and treating affected individuals at the point when defects occur (Brasseur-Daudruy et al., 2011). Therefore, fully understanding the molecular pathomechanisms leading to the complex disease phenotypes of these disorders is imperative to future efforts in diagnostic testing and therapeutic intervention.
While this study primarily focused on a model
Conclusions
In summary, herein we reported that RPTPζ/phosphacan is hypoglycosylated in animal models of MEB lacking functional POMGnT1. These results suggest that RPTPζ/phosphacan is a significant substrate for protein O-mannosyl glycosylation in vivo, displaying the loss of several carbohydrate epitopes including HNK-1. Additionally, this work identifies the epitopes of several previously uncharacterized carbohydrate antibodies against RPTPζ/phosphacan as likely detecting O-mannosyl-linked glycans. While
Acknowledgements
The authors would like to thank Peng Zhang for animal genotyping and Wendi Burnette for technical assistance.
This work was funded by NIH/NINDS Grant #NS069660 to R.T.M and Grant #HD060458 and NS066582 to H.H.
Glossary
- Receptor protein tyrosine phosphatase ζ (RPTPζ)/phosphacan
- A chondroitin sulfate proteoglycan with CNS-enriched expression that is encoded by the PTPRZ1 gene. The full-length form of the protein functions as a receptor protein tyrosine phosphatase, while alternative splicing gives rise to a secreted form called phosphacan. While the full length- form of the receptor (RPTPζ) and secreted variant (phosphacan) are considered to be long-isoforms, short-isoforms of each variant have also been
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