OPN (osteopontin) is a multiphosphorylated extracellular glycoprotein, which has important roles in bone remodelling, inflammation and cancer metastasis. OPN regulates cell spreading and adhesion primarily through its association with several integrins such as αvβ3, and its phosphorylation affects these processes. However, the mechanism by which OPN O-glycosylation affects these processes is not completely understood. In the present study, we demonstrated that OPN O-glycosylation self-regulates its biological activities and also affects its phosphorylation status. We prepared two recombinant OPNs, WT (wild-type)-OPN and mutant OPN (ΔO-OPN), which lacks five O-glycosylation sites at a threonine/proline-rich region. O-glycan defects in OPN increased its phosphorylation level, as observed by dephosphorylation assays. Moreover, compared with WT-OPN, ΔO-OPN exhibited enhanced cell spreading and adhesion activities and decreased associations with β1 integrins. This suggested that defects in O-glycans in OPN altered these activities, and that β1 integrins have a less important role in adhesion to ΔO-OPN. The cell-adhesion activity of dephosphorylated ΔO-OPN was higher than the cell-adhesion activities of ΔO-OPN and dephosphorylated WT-OPN. This suggested that some of the phosphorylation in ΔO-OPN caused by O-glycan defects and O-glycans of OPN suppressed the OPN cell-adhesion activity. Thus functional activities of OPN can be determined by the combined glycosylation and phosphorylation statuses and not by either status alone.

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr¹³⁴/Thr¹³⁸/Thr¹⁴³/Thr¹⁴⁷/Thr¹⁵²) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr¹³⁴/Thr¹³⁸ or Thr¹⁴³/Thr¹⁴⁷/Thr¹⁵² had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC–MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

Glycosylation is a cell type-specific post-translational modification that can be used for biomarker identification in various diseases. Aim of this study is to explore glycan-biomarkers on transferrin (Tf) for Alzheimer’s disease (AD) in cerebrospinal fluid (CSF). Glycan structures of CSF Tf were analyzed by ultra-performance liquid chromatography followed by mass spectrometry. We found that a unique mannosylated-glycan is carried by a Tf isoform in CSF (Man-Tf). The cerebral cortex contained Man-Tf as a major isofom, suggesting that CSF Man-Tf is, at least partly, derived from the cortex. Man-Tf levels were analyzed in CSF of patients with neurological diseases. Concentrations of Man-Tf were significantly increased in AD and mild cognitive impairment (MCI) comparing with other neurological diseases, and the levels correlated well with those of phosphorylated-tau (p-tau), a representative AD marker. Consistent with the observation, p-tau and Tf were co-expressed in hippocampal neurons of AD, leading to the notion that a combined p-tau and Man-Tf measure could be a biomarker for AD. Indeed, levels of p-tau x Man-Tf showed high diagnostic accuracy for MCI and AD; 84% sensitivities and 90% specificities for MCI and 94% sensitivities and 89% specificities for AD. Thus Man-Tf could be a new biomarker for AD.

Iron plays many important roles in the brain, including involvement in myelination, neurotransmission and electron transfer in the respiratory chain. Transferrin (Tf), an iron transporter, is mainly biosynthesized in the liver, but can also be biosynthesized in the brain; i.e., by oligodendrocytes and the choroid plexus, a cerebrospinal fluid (CSF) producing tissue. The CSF contains two Tf isoforms, brain-type Tf and serum-type Tf, which differ in their glycan structures. Brain-type Tf is uniquely glycolsylated with biantennary asialo- and agalacto-complex type N-glycans that carry bisecting β1,4-GlcNAc and core α1,6-Fuc. The glycans of serum-type Tf in the CSF are similar to those of Tf in serum. Biochemical analyses reveal that the apparent molecular size of brain-type Tf is smaller than that of serum-type Tf, and that hydrophobic patches are exposed on brain-type Tf as demonstrated by hydrophobic probe binding studies. We found that brain-type Tf levels were decreased in idiopathic normal pressure hydrocephalus, in which CSF production is suspected to decrease, while brain-type Tf increased in spontaneous intracranial hypotension, in which CSF production is suspected to increase. These results suggest that brain-type Tf could be a biomarker of altered CSF production.

Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system that predominantly affects the optic nerves and spinal cord. Although NMO has long been considered a subtype of multiple sclerosis (MS), the effects of interferon-β treatment are different between NMO and MS. Recent findings of NMO-IgG suggest that NMO could be a distinct disease rather than a subtype of MS. However, the underlying molecular mechanism of NMO pathology remains poorly understood. OPN in the cerebrospinal fluid and brain of patients with NMO and with MS, as well as of patients with other neurologic disease/idiopathic other neurologic disease was examined using Western blotting, ELISA, immunohistochemistry and Boyden chamber. Here we show that osteopontin is significantly increased in the cerebrospinal fluid of NMO patients compared with MS patients. Immunohistochemical analyses revealed that osteopontin was markedly elevated in the cerebral white matter of NMO patients and produced by astrocytes, neurons, and oligodendroglia as well as infiltrating macrophages. We also demonstrate that the interaction of the cerebrospinal fluid osteopontin in NMO patients with integrin αvβ3 promoted macrophage chemotaxis by activating phosphoinositide 3-kinase and MEK1/2 signaling pathways. These results indicate that osteopontin is involved in NMO pathology. Thus therapeutic strategies that target osteopontin signaling may be useful to treat NMO.