Identification Of The Crystal Structure Of The Lamprey LIP Protein And Activity Identification Of Related Site Mutations

Lamprey belongs to the primitive lamprey vertebrates and is an important model organism for studying the origin and evolution of immunity.Our previous study found that Lamprey immune protein?LIP?is a cytotoxic protein in the lamprey.At present,we have a deeper understanding of the information and functions of this unique protein.LIP exhibits strong cytocidal activities against human cancer cells,in contrast to have no effect on normal cells.In addition,many results confirm that LIP protein induce remarkable morphological changes in tumor cells and disrupt the cell membrane and organelle,finally lead to tumor cells death.In summary,our data can provide an experimental basis for cancer treatment of LIP as marine drugs.Native LIP proteins have a variety of modifications,but the specific information is still unknown.A variety of different modifications of the LIP protein were detected by DIA proteomic quantification techniques,with N-glycosylation residues in Asn286and O-glycosylation residues in Ser295 for the detection of glycosylation sites.Both of these amino acids were mutated to Ala as mutant fifth LIP^N286A286A and mutant sixth LIPS295A.In order to deeply explore the spatial structure of LIP,the LIP protein was crystallized.After data collection,phase analysis and analysis of the data,the LIP protein structure was obtained as P4₃2₁2 space group with a resolution of 44.5².25?.The hydrogen bond network of sucrose or mannose stably bound in LIP was found to be located at the most central position of binding to the ligand,and the amino acid residue Asp was mutated to Ala as the first mutant LIP^D135A.LIP crystal structure analysis also showed that it has two domains,Jacalin-like and Aerolysin.After LIP was homologously modeled based on the similar structure of Dln1,it was found that Met158,Phe229 and Pro163 and Phe227 residues on the two-body interaction surface were possible to form hydrogen bond,therefore,these four amino acids were mutated to cysteines,respectively,and it is desirable to form a disulfide bond in the mutation as the second mutant LIP^M158C-F229C158C-F229C and the third mutant LIP^P163C-F227C.Amino acid sequence alignment and hydrophobicity analysis indicated that the range of amino acid from Phe209 to Gly232 of LIP could form two amphipathic?chains containing hydrophobic hydrophilic residues,and this amino acid chain was removed as the fourth mutant LIP^{Ser212-Ala238}.The LIP protein and the six mutant proteins were detected by circular dichroism and fluorescence spectroscopy,and they were all secondary structures mainly composed of?-sheets.Aside from LIP^Ser212-Ala238er212-Ala238 more hydrophilic,there was no particularly large difference in structures.The mutants LIP^D135A,LIP^M158C-F229C158C-F229C and LIP^P163C-F227C163C-F227C had no cytocidal effect on cancer cells without significant change in the secondary structure,and could not specifically bind to the cell membrane surface,providing the forming of two interacting surfaces each other.The hydrogen-bonded Met158,Phe229 and Pro163,Phe227 residues and the Asp135 residue at the center of the hydrogen bond network play an important role in LIP-specific recognition and killing of cancer cells.For LIP^S295A295A mutant,the removal of the O-glycosylation residue at the Ser295 site could not affect the structure and function of the LIP protein;the mutant LIP^N286A286A retained this cytocidal function and specifically bound to the cell membrane,indicating that the removal of N-glycosylation residues at the Asn286 site can greatly attenuate the effect of killing tumor cells without affecting cell membrane binding.In summary,LIP and LIP^S295A295A proteins showed significant killing effect on MCF-7 cells through LDH detection.Based on the results of immunofluorescence detection,LIP,LIP^N286A286A and LIP^S295A295A proteins were localized on the cell membrane of HeLa cells,westhern blot assays of multimers on the cell membrane showed results identical to those of the immunofluorescence.It is worth noting that the LIP^N286A286A mutant protein has not only obvious killing effect on MCF-7 cells,but also can specifically bind to the cell membrane surface.Super-resolution microscopy results indicate that LIP^N286A286A mutant protein and probe of membrane-labeled dye Cholera Toxin Subunit B Conjugated with Alexa 555 does not completely overlap on the cell membrane surface,demonstrating the development of the LIP^N286A286A mutant protein as a novel cell membrane dye.