Recently, the enzyme family of oleate hydratases (OHs: EC 4.2.1.53) has gained increasing scientific and economic interest, as these FAD‐binding bacterial enzymes do not require cofactor recycling and possess high thermal and pH stability. Their products, hydroxy fatty acids, are used in specialty chemical applications including surfactant and lubricant formulations. The “oleate hydratase engineering database”, established by Schmid et al. (2017), divides all OHs into 11 families (HFam1 to 11). To date, only two crystal structures of homodimeric OHs from the families HFam2 and HFam11 have been reported. In this study, we biophysically characterized an OH belonging to the HFam3 family, originating from the marine bacterium Rhodococcus erythropolis, for the first time. The crystal structure revealed that this new OH (OhyRe) surprisingly is a monomer in its active form. This particular feature provides new avenues for enzyme engineering and recycling through immobilization.
Tight junction (TJ) formation is vital for epidermal barrier function. We aimed to specifically manipulate TJ barriers in the reconstructed human epidermis (RHE) by claudin‐1 and ‐4 knockdown (KD) and by claudin‐binding fusion proteins of glutathione S‐transferase and modified C‐terminal fragments of Clostridium perfringens enterotoxin (GST‐cCPE). Impedance spectroscopy and tracer permeability imaging were employed for functional barrier assessment and investigation of claudin contribution. KD of claudin‐1, but not claudin‐4, impaired the paracellular barrier in vitro. Similarly, claudin‐binding GST‐cCPE variants weakened the paracellular but not the stratum corneum barrier. Combining both TJ targeting methods, we found that claudin‐1 targeting by GST‐cCPE after claudin‐4 KD led to a marked decrease in paracellular barrier properties. Conversely, after claudin‐1 KD, GST‐cCPE did not further impair the barrier. Comparison of GST‐cCPE variants with different claudin‐1/claudin‐4 affinities, NHS‐fluorescein tracer detection, and immunostaining of RHE paraffin sections showed that GST‐cCPE variants bind to extrajunctional claudin‐1 and ‐4, which are differentially distributed along the stratum basale–stratum granulosum axis. GST‐cCPE binding blocks these claudins, thereby specifically opening the paracellular barrier of RHE. The data indicate a critical role for claudin‐1 in regulating paracellular permeability for ions and small molecules in the viable epidermis. Claudin targeting is presented as a proof‐of‐concept for precise barrier modulation.
Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec–glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.
Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.
The Cover Feature displays the crystal structure of the oleate hydratase OhyRe from Rhodococcus erythropolis, structurally and biochemically characterized for the first time, which catalyzes the hydration and dehydration of oleic acid. In their Full Paper, J. Lorenzen, R. Driller et al. elucidated the crystal structure of OhyRe, which revealed that the active form of OhyRe is a monomer, in contrast to other known oleate hydratase structures. Mutation of key residues resulted in decreased enzymatic activity, indicating a distinct reaction mechanism among the family members of hydratases. More information can be found in the Full Paper by J. Lorenzen, R. Driller et al. on page 407 in Issue 2, 2018 (DOI: 10.1002/cctc.201701350).
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