In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease.

As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.

O-glycosylation on cerebrospinal fluid and plasma apolipoprotein E differs in the lipid-binding domain.

The O-glycoprotein apolipoprotein E (APOE), the strongest genetic risk factor for Alzheimer's disease, associates with lipoproteins. Cerebrospinal fluid (CSF) APOE binds only high-density lipoproteins (HDLs), while plasma APOE attaches to lipoproteins of diverse sizes with binding fine-tuned by the C-terminal loop. To better understand the O-glycosylation on this critical molecule and differences across tissues, we analyzed the O-glycosylation on APOE isolated from the plasma and CSF of aged individuals. Detailed LC-MS/MS analyses allowed the identification of the glycosite and the attached glycan and site occupancy for all detectable glycosites on APOE and further three-dimensional modeling of physiological glycoforms of APOE. APOE is O-glycosylated at several sites: Thr8, Thr18, Thr194, Ser197, Thr289, Ser290 and Ser296. Plasma APOE held more abundant (20.5%) N-terminal (Thr8) sialylated core 1 (Neu5Acα2-3Galß1-3GalNAcα1-) glycosylation compared to CSF APOE (0.1%). APOE was hinge domain glycosylated (Thr194 and Ser197) in both CSF (27.3%) and plasma (10.3%). CSF APOE held almost 10-fold more abundant C-terminal (Thr289, Ser290 and Ser296) glycosylation (36.8% of CSF peptide283-299 was glycosylated, 3.8% of plasma peptide283-299), with sialylated and disialylated (Neu5Acα2-3Galß1-3(Neu5Acα2-6) GalNAcα1-) core 1 structures. Modeling suggested that C-terminal glycosylation, particularly the branched disialylated structure, could interact across domains including the receptor-binding domain. These data, although limited by sample size, suggest that there are tissue-specific APOE glycoforms. Sialylated glycans, previously shown to improve HDL binding, are more abundant on the lipid-binding domain of CSF APOE and reduced in plasma APOE. This indicates that APOE glycosylation may be implicated in lipoprotein-binding flexibility.

Synthetic standard aided quantification and structural characterization of amyloid-beta glycopeptides enriched from cerebrospinal fluid of Alzheimer's disease patients.

An early pathological hallmark of Alzheimer's disease (AD) is amyloid-ß (Aß) deposits in the brain, which largely consist of up to 43 amino acids long Aß peptides derived from the amyloid precursor protein (APP). We previously identified a series of sialylated Tyr-10 O-glycosylated Aß peptides, 15-20 residues long, from human cerebrospinal fluid (CSF) and observed a relative increase of those in AD vs non-AD patients. We report here on the synthesis and use of an isotopically double-labeled Aß1-15 glycopeptide, carrying the core 1 Galß3GalNAcα1-O-Tyr-10 structure, to (1) identify by HCD LC-MS/MS the definite glycan core 1 structure of immunopurified and desialylated Aß glycopeptides in human CSF and to (2) establish a LC-MS/MS quantification method for desialylated Aß1-15 (and Aß1-17) glycopeptides and to (3) compare the concentrations of these Aß glycopeptides in CSF from 20 AD patients and 20 healthy controls. Although we unambiguously identified the core 1 structures and Tyr-10 attachment sites of the glycopeptides, we did not observe any quantitative differences, determined through both peptide and oxonium ion fragments, of the desialylated Aß1-15 or Aß1-17 glycopeptides between the AD and non-AD group. The new quantitative glycoproteomic approach described, using double-labeled glycopeptide standards, will undoubtedly facilitate future studies of glycopeptides as clinical biomarkers but should also embrace sialylated Aß standards to reveal specific sialylation patterns of individual Aß glycopeptides in AD patients and controls.

Immunoluminometric assay for copeptin measurement in cerebrospinal fluid: Technical aspects and pilot study.

BACKGROUND: Copeptin acts as surrogate marker under stress stimuli, as well as an outcome predictor based on serum or plasma concentration in patients suffering intracranial hemorrhage, aneurysmal subarachnoid hemorrhage (aSAH), and stroke. The aim of this study was to establish a method for quantification of copeptin levels in cerebrospinal fluid (CSF) and to demonstrate its clinical applicability in patients following aSAH. METHODS: This assay was validated for CSF samples using a commercial immunoluminometric assay (IMLA). For the control group (10 patients), CSF copeptin levels were determined in patients without signs of acute neurological diseases and who underwent a diagnostic lumbar puncture. The pilot cohort included calculation of copeptin levels in CSF and in serum of patients following aSAH. RESULTS: The control group had CSF copeptin levels lower than 0.78 pmol/L-1. Among patients with aSAH, CSF copeptin values had a mean of 20.1 pmol/L-1 and serum copeptin concentrations had a mean of 61.39 pmol/L-1. CONCLUSIONS: This assay provides to best of our knowledge for the first time initial ranges values of CSF copeptin for patients without acute neurological disease and in patients with aSAH. Thus, it opens new doors to develop further calculations and relationships between diseases biomarker and outcome prediction.

Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

Sialic acid capture-and-release and LC-MS(n) analysis of glycopeptides.

Extracellular glycoproteins frequently carry terminal sialic acids on their N-linked and/or O-linked glycan structures. In this chapter a sialic acid specific capture-and-release protocol for the enrichment of N- and O-glycopeptides originating from glycoproteins in complex biological samples is described. The enriched glycopeptides are subjected to reversed phase liquid chromatography (LC) interfaced with electrospray ionization and multistage tandem mass spectrometry (MS(n)). The glycopeptide precursor ions are fragmented by collision-induced dissociation (CID) for analysis of the glycan parts in the MS(2) spectra. Further fragmentation (i.e., MS(3)) of deglycosylated peptide ions results in peptide backbone fragmentation, which is used in protein database searches to identify protein sequences. For O-glycopeptides the use of both CID and electron capture dissociation (ECD) fragmentation of the peptide backbone with intact glycans still attached are used to pinpoint the glycosylation sites of glycopeptides containing several Ser/Thr residues. The step-by-step protocols for fragmentation analyses of O- and N-glycopeptides enriched from human cerebrospinal fluid are described.

CNS penetration of the opioid glycopeptide MMP-2200: a microdialysis study.

Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson's disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood-brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/µ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10 mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80 min). The detected level at the end of the experiment (around 250 pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood-brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS.

Evaluation of intraventricular teicoplanin for the treatment of neurosurgical shunt infections.

Seven patients with staphylococcal neurosurgical shunt infections were treated with intraventricular teicoplanin. Two infants received 5 mg/d, three patients received 20 mg/d, and two patients received 20 mg every other day. Six of these patients also received intravenous antibiotics. Three patients had infections caused by methicillin-susceptible Staphylococcus epidermidis, and one patient had an infection caused by methicillin-resistant S. epidermidis. Three patients were infected with Staphylococcus aureus (one with a methicillin-resistant strain and two with methicillin-susceptible strains). The mean duration of intraventricular therapy was 16 days. Sterilization of cerebrospinal fluid (CSF) was obtained after an average of 4.4 days. All patients were cured both clinically and microbiologically. No significant adverse effects were observed in any patients. Penetration of teicoplanin into the CSF after intravenous administration was poor. However, after intraventricular administration, high and prolonged peak and trough levels of teicoplanin were detected in the CSF. Bactericidal activity of the CSF was remarkable, exceeding the 1:8 dilution in the majority of the cases. The alternate-day schedule of intraventricular administration of teicoplanin was as effective as the once-daily regimen.

Influence of mannitol on the penetration of teicoplanin into infected CSF of experimental Staphylococcus aureus meningitis of rabbits.

We investigated the influence to mannitol injections on the teicoplanin penetration into CSF in experimental bacterial meningitis of rabbits. Experimental bacterial meningitis was obtained by intracisternal inoculation of 10(7) cfu of methicillin resistant Staphylococcus aureus. The experimental bacterial meningitis was controlled 18 h later by cisternal puncture. At this time (T0) mannitol (solution for injection 20%) was injected (bolus), at a dosage of 3 ml/kg, into the carotid arteria. Immediately after the previous bolus, teicoplanin was administered through the same line over 5 min, at a dosage of 5 mg/kg. Cerebrospinal fluid was obtained 30 min and 2 h after injection was completed, and serum samples were obtained at the same time. Results were (mg/l) in cerebrospinal fluid: (Table; see text) (* p less than 0.05 comparing the two regimens at T0 + 2 h, and comparing regimen teicoplanin + mannitol infusion at T0 + 30 sec and + 2 h). This is very promising for the treatment of methicillin resistant Staphylococcus aureus bacterial meningitits and should support further investigations.

Efficacy of teicoplanin in experimental group B streptococcal bacteremia and meningitis.

We evaluated the activity of teicoplanin against a type-III group B streptococcal strain in vitro and in vivo and compared the results with those of penicillin G. In vitro, the minimal inhibitory and minimal bactericidal concentrations of teicoplanin were 2- to 4-fold greater than those of penicillin G. In vivo studies were carried out with an experimental bacteremia and meningitis model in newborn rats. Eighty-one infected animals were randomized to receive teicoplanin 5, 10 or 20 mg/kg, twice daily, or penicillin G 50 or 200 mg/kg, twice daily, or saline (0.05 ml), twice daily. The mean serum levels of teicoplanin were maintained above 100 X the minimal bactericidal concentration for 7-8 h even with a dose of 5 mg/kg. The mean penetration of teicoplanin into the cerebrospinal fluid was estimated as 2.4-8.2% of those of concomitant levels in serum. The overall efficacy of teicoplanin was similar to that of penicillin G as judged by mortality rates. However, two bacteremic animals which were free of meningitis at the beginning of therapy developed this complication during 4 days of teicoplanin therapy, in contrast with none in the penicillin group. Further studies are needed to understand the reason(s) for these failures with teicoplanin therapy.

Teicoplanin in the therapy of staphylococcal neuroshunt infections.

Three patients with staphylococcal infection of neuroshunt, two with ventriculoperitoneal and one with external ventricular derivation, were treated with teicoplanin given intravenously (iv) or intraventricularly (ivt), two patients were adults, while the third was an infant. The causative organisms were methicillin-sensitive Staphylococcus epidermidis (MSSE) in the first two cases and methicillin-sensitive Staphylococcus aureus (MSSA) in the paediatric patient. Patient No. 1 was given teicoplanin iv (400 mg/day) and subsequently ivt (20 mg/day). CSF teicoplanin levels were always below 1 microgram/ml during the first part of the treatment; after the institution of ivt therapy, drug levels ranged from 20 to 35 micrograms/ml (mean 26.4). The patient was cured after 20 days of a combination treatment (ivt teicoplanin plus rifampin, 900 mg/day, iv). Patient No. 2 was given teicoplanin 400 mg/day, iv, for two weeks; its low levels in CSF (always below 0.5 microgram/ml) accounted for the failure to eradicate the MSSE. A new regimen with teicoplanin, 20 mg/day plus netilmycin, 15 mg/day, both ivt, cured the patient in two weeks. Teicoplanin levels in the CSF ranged from 12.5 to 33 micrograms/ml (mean 18.3). Patient No. 3, an 8-month-old infant, was placed on teicoplanin, 5 mg/day ivt plus chloramphenicol, 180 mg/day iv, after several other antibiotic trials had failed to eradicated a MSSA infection of his external ventricular derivation. The therapy cleared the infection in one week (CSF teicoplanin levels: 30 to 38, mean 34 micrograms/ml). Teicoplanin (iv or ivt) was well tolerated without any significant side-effects; its role in the treatment of staphylococcal neuroshunt infections deserves further studies.