A Nanoplasmonic Assay for Point-of-Care Detection of Mannose-Binding Lectin in Human Serum.

Mannose-binding lectin (MBL) activates the complement system lectin pathway and subsequent inflammatory mechanisms. The incidence and outcome of many human diseases, such as brain ischemia and infections, are associated with and influenced by the activity and serum concentrations of MBL in body fluids. To quantify MBL levels, tests based on ELISA are used, requiring several incubation and washing steps and lengthy turnaround times. Here, we aimed to develop a nanoplasmonic assay for direct MBL detection in human serum at the point of care. Our assay is based on gold nanorods (GNRs) functionalized with mannose (Man-GNRs) via an amphiphilic linker. We experimentally determined the effective amount of sugar linked to the nanorods' surface, resulting in an approximate grafting density of 4 molecules per nm2, and an average number of 11 to 13 MBL molecules binding to a single nanoparticle. The optimal Man-GNRs concentration to achieve the highest sensitivity in MBL detection was 15 µg·mL-1. The specificity of the assay for MBL detection both in simple buffer and in complex pooled human sera was confirmed. Our label-free biosensor is able to detect MBL concentrations as low as 160 ng·mL-1 within 15 min directly in human serum via a one-step reaction and by using a microplate reader. Hence, it forms the basis for a fast, noninvasive, point-of-care assay for diagnostic indications and monitoring of disease and therapy.

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF121, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF121, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF121 and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF121. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.