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  • In summary we obtained and type

    2022-08-04

    In summary, we obtained - and -type of linaclotide by protein chemical synthesis and acquired the X-ray crystal structure of linaclotide for the first time through racemic crystallization technique. The structure of linaclotide forms a compact spatial structure containing three- turns through three intramolecular disulfide bonds, which has a similar spatial arrangement to heat-stable enterotoxin of (ST mimic). We anticipated that the X-ray crystal structure of linaclotide provides the basis for the optimization and improvement of the activity of linaclotide. Our work also highlights the power of chemical protein synthesis in obtaining peptide drugs for biochemical and structural studies. Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC No. 21572043) and the Fundamental Research Funds for the Central Universities (No. PA2017GDQT0021).
    Introduction Vertebrate rod and cone photoreceptor Cathepsin K Activity Fluorometric Assay Kit receptor respond to light by a change of the membrane potential. A light-triggered enzymatic cascade leads to the hydrolysis of the intracellular messenger guanosine-3′, 5′-cyclic monophospate (cGMP), which is synthesized by membrane bound guanylate cyclases [1]. While rod cells operate at low light intensities, cone cells are sensitive at higher levels of ambient illumination and maintain their responsiveness at fluctuating background light [2]. Adaptation to different background light intensities is mediated by an operative network of photoreceptor proteins and a change of intracellular calcium concentration. Calcium sensor proteins detect these changes and regulate their targets in a calcium dependent manner [1], [2], [3], [4], [5], [6]. Among them are the recoverins and the guanylate cyclase-activating proteins (GCAPs) [3], [4], [5] that belong to the family of neuronal calcium sensor proteins [6], [7]. Mammalian GCAP1 and GCAP2 are expressed in rods and cones and regulate membrane bound guanylate cyclases in a calcium-dependent way, but differ in their biochemical and physiological properties [8], [9], [10]. A larger variety of GCAPs was recently detected in retina of adult zebrafish [11], among them are four isoforms (zGCAP3, zGCAP4, zGCAP5 and zGCAP7) specifically expressed in cones [11], [12]. However, almost nothing is known about guanylate cyclases in the zebrafish retina as possible targets of zGCAPs. Only three mRNA sequences that code for putative sensory guanylate cyclases (zGCs) had been deposited in the GenBank database (gucy2f, gc2, gc3), but the cloning of the full-length sequences and a characterization of their molecular properties has not been reported so far. On the molecular level excitation and adaptation in cones are less well understood than in rods. But recently, the zebrafish has attained increasing interest as a model organism to study cone vision. Zebrafish larvae respond to visual stimuli as early as 3 days post fertilization (3 dpf) and electroretinogram (ERG) recordings indicate retinal function in larvae at 4 dpf (see for example [13], [14], [15], [16]). The visual system of the zebrafish contains five photoreceptor cell types: rods, double cones consisting of a long and a short member (DC), long single cones (LSC) and short single cones (SSC). These five cell types express a total of nine opsin genes (one rod opsin and eigth cone opsin genes). The absorption characteristics of the opsins in cone cells indicate that zebrafish posses colour vision similar to that of human [17], [18].
    Materials and methods
    Results
    Discussion Membrane bound guanylate cyclases in vertebrate photoreceptor cells respond to changing cytoplasmic Ca2+-concentrations and thereby maintain a dynamic synthesis of cGMP in the cell. While mammals express only 2 photoreceptor specific GCs and 2–3 regulatory GCAPs, teleosts have a larger diversity of GCs and GCAPs. In the present study we report on the temporal and spatial expression of membrane bound sensory GC isoforms in the larval and adult zebrafish retina. Zebrafish larvae respond to visual stimuli as early as 3 dpf and electroretinogram recordings indicate retinal function in larvae at 4 dpf (see for example [13], [14], [15], [16]). Thus, expression of zGCs coincided with the onset of visual function at 3–4 dpf. Further, three cone specific zGCAPs were also expressed at the same stage of retina maturation.