Investigating LGALS3BP/90 K glycoprotein in the cerebrospinal fluid of patients with neurological diseases

Abstract

Galectin-3 binding protein (LGALS3BP or 90 K) is a secreted glycoprotein found in human body fluids. Deregulated levels were observed in cancer and infection and its study in neurological diseases is more recent. Here, we have investigated 90 K from human cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS, n = 35) and other neurological diseases (n = 23). CSF was fractionated by ultrafiltration/size-exclusion chromatography (SEC) and eluted fractions were analysed by complementary techniques including immunoblotting, electron microscopy and nano-liquid chromatography-tandem mass spectrometry. A fraction of 90 K appeared as nanoparticles of irregular shape with heterogeneous dimensions of 15-60 nm that co-eluted with extracellular vesicles in SEC. Median levels of 90 K quantified by ELISA were not different between ALS patients (215.8 ng/ml) and controls (213.3 ng/ml) in contrast with the benchmark biomarker for ALS phosphoneurofilament heavy chain (1750 and 345 pg/ml, respectively). A multiregression model supported age is the only independent predictor of 90 K level in both groups (p < 0.05). Significant correlation was found between 90 K levels and age for the ALS group (r = 0.366, p = 0.031) and for all subjects (r = 0.392, p = 0.003). In conclusion, this study unveils the presence of 90 K-containing nanoparticles in human CSF and opens novel perspectives to further investigate 90 K as potential aging marker. Galectin-3-binding protein (LGALS3BP, also known as Mac-2BP or tumor-associated antigen 90 K) is a secretory protein with 567 amino acid residues 1. It is heavily N-glycosylated with seven glycosylation sites 2. In early studies 90 K was purified from supernatants of tumor cells where it appeared as large molecular mass complexes 3. Purified full-length recombinant glycoprotein from human embryonic kidney cells was found to self-associate into non-covalent oligomers of 1000-1500 kDa that appeared as ring-like structures and other shapes by electron microscopy. Recombinant 90 K also associated with collagens IV, V and VI, fibronectin and nidogen, it mediated cell adhesion and it was present in the extracellular matrix of mouse tissues 4. 90 K was visualized by electron microscopy in nanoparticles of irregular shape found in extracellular vesicle (EV) fractions from tumor cells in culture 5. More recently, asymmetric flow field-flow fractionation revealed novel nanoparticles from supernatants of tumor cells in culture termed exomeres, which contained 90 K 6. Cells from the human body as well as cells in culture release EV to the surroundings. EV include two major groups, exosomes of endosomal origin and microvesicles that derive from the plasma membrane of cells. EV can be found in body fluids, such as blood 7 or CSF 8-11 and constitute promising tools in disease diagnosis 12. In view of EV heterogeneity several isolation techniques have been used including ultracentrifugation and size-exclusion chromatography (SEC) 12 .

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…This fact has popularized the use of SEC amongst its competitors for blood-based exosome-associated biomarker discovery research [ 83 , 89 ]. SEC has been used successfully to isolate, purify and enrich exosomes from a variety of biological fluids including plasma [ 20 , 21 , 22 , 23 , 24 , 25 ], serum [ 26 , 27 , 28 ], bovine and human milk [ 29 , 30 ], urine [ 31 , 32 , 33 , 34 ], saliva [ 35 , 36 ], tears [ 35 ], cerebrospinal fluid [ 37 , 38 ], synovial fluid [ 39 ], nasal lavage [ 41 ], seminal fluid, [ 40 ] and stromal vascular fraction (SVF) from adipose tissue [ 42 , 43 ] ( Table 2 ). Recent findings reported at the ISEV Virtual Conference 2020 suggest SEC is superior to other techniques in isolation of pure exosomes from human body fluids (unpublished findings).…”

“…Since a single exosome isolation technique cannot reach optimal yield and purity, coupling SEC with other isolation methods including dUC, UF, or PEG-based retrieval can precipitate intact, highly purified exosomes in a reproducible manner. However, the limitations of these approaches as detailed above will then accompany the chimeric protocol [ 32 , 37 , 44 , 49 , 90 , 101 , 102 , 103 ].…”

Expression of recombinant cardosin B in tobacco BY2 cells: an alternative system for the production of active milk clotting enzymes

“…Afterward, the gel was stained with BlueSafe (NZYTech, Portugal) and protein bands were cut out using a scalpel blade. Protein identification was performed by mass spectrometry as previously described 35 , with exception of the Uniprot database used for protein identification. Gel bands were reduced in 10 mM DTT (Sigma) for 40 min at 56⁰C, and alkylated in 55 mM iodoacetamide (Sigma) for 30 min in the dark.…”

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…This fact has popularized the use of SEC amongst its competitors for blood-based exosome-associated biomarker discovery research [52], [58]. SEC has been used successfully to isolate, purify and enrich exosomes from a variety of biological fluids including plasma [68]- [73], serum [74]- [76], bovine and human milk [77], [78], urine [79]- [82], saliva [83], [84], tears [83], cerebrospinal fluid [85], [86], synovial fluid [87], nasal lavage [89], seminal fluid [88] and stromal vascular fraction (SVF) from adipose tissue [63], [64] (Table 3). Recent findings reported at the ISEV Virtual Conference 2020 suggest SEC is superior to other techniques in isolation of pure exosomes from human body fluids (unpublished findings).…”

“…Since a single exosome isolation technique cannot reach optimal yield and purity, coupling SEC with other isolation methods including dUC, UF, or PEG-based retrieval can precipitate intact highly purified exosomes in a reproducible manner. However, the limitations of these approaches as detailed above will then accompany the chimeric protocol [59], [80], [85], [90], [95], [102]- [104].…”