Depletion of highly abundant proteins from human cerebrospinal fluid: a cautionary note

Affinity-based techniques, both for enrichment or depletion of proteins of interest, suffer from unwanted interactions between the bait or matrix material and molecules different from the original target. This effect was quantitatively studied by applying two common procedures for the depletion of albumin/gamma immunoglobulin to human cerebrospinal fluid. Proteins of the depleted and the column-bound fraction were identified by mass spectrometry, employing 18O labeling for quantitation of their abundance. To different extents, the depletion procedures caused the loss of proteins previously suggested as biomarker candidates for neurological diseases. This is an important phenomenon to consider when quantifying protein levels in biological fluids

Removal of albumin and immunoglobulins from canine cerebrospinal fluid using depletion kits: a feasibility study.

International audienceBACKGROUND: Highly abundant proteins in biological fluids such as serum or cerebrospinal fluid (CSF) can hinder the detection of proteins in lower abundance, e.g., potential biomarkers. Commercial products are available for the depletion of albumin and immunoglobulins (Igs), although most of these kits have not been validated for dog samples. The present study therefore examines the use of different types of depletion kits for dog CSF. FINDINGS: Three kits, with different mechanisms for the depletion of albumin and Igs, were tested with dog CSF specimens. One product significantly decreased the amount of albumin; with all kits, IgG was less efficiently removed than albumin. Mass spectrometry of the fractions eluted from the depletion columns revealed considerable co-depletion of other CSF proteins. CONCLUSIONS: A commercially available depletion kit was identified which depletes albumin and (to a lower extent) immunoglobulins from dog CSF. However, the limited efficacy and the concomitant loss of other proteins from the sample should be taken into account when using this product

Additional file 2: Figure S1. of Depletion of highly abundant proteins from human cerebrospinal fluid: a cautionary note

Protein fractions obtained by depletion of human cerebrospinal fluid (CSF), separated by sodium dodecyl sulfate gel electrophoresis and stained by Coomassie Brilliant Blue G250. (Protein marker, M; CSF undepleted, C; depleted (=flow-through) fraction, FT; column-bound (=eluate) fraction, E); protein load, 5 μg/lane. (PDF 71 kb

Redox regulation of cell contacts by tricellulin and occludin: redox-sensitive cysteine sites in tricellulin regulate both tri- and bicellular junctions in tissue barriers as shown in hypoxia and ischemia

Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs: tricellulin, occludin, marvelD3) are thought to be regulatory relevant. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. Aim: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys-sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. Results: Ischemia, hypoxia and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys-sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. Innovation: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox-dependently; a structural model is proposed. Conclusions: TAMPs' ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox-sensor and sealing element at 3-cell contacts and may compensate as redox-mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox-regulator for ischemia-related alterations

Redox-Sensitive Structure and Function of the First Extracellular Loop of the Cell–Cell Contact Protein Claudin-1: Lessons from Molecular Structure to Animals

The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. AIMS: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. RESULTS: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a β-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. INNOVATION: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding β-sheet, stabilized by a shielded and redox-sensitive disulfide bond. CONCLUSION: Since the β-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers