Molecular Cloning And Functional Characterization Of Two C-type Lectins Of The Tubeworm From A Deep-sea Hydrothermal Vent

C-type lectins(CTLs)are proteins capable of recognizing and binding carbohydrates.CTLs participate in numerous cellular processes,including cell-cell interaction,adhesion,cellular signaling,and innate immunity.Tubeworm is the representative species in many deep-sea hydrothermal vents.Currently,the immune system of hydrothermal tubeworms is essentially unknown.To explore the functional characterization of immune system of deep-sea tubeworms,we focus on the function of CTLs from tubeworms collected from a hydrothermal vent in Pacmanus.In this study,tubeworm Alaysia sp.were collected from a hydrothermal vent in Pacmanus by the vessel KEXUE.We identified and characterized two CTLs(Lec1 and Lec2)from Alaysia sp..Sequence analysis showed that Lec1 and Lec2 possess the typical CTL domain,they share 30.6%identity in overall sequence and share low sequence identities(10.8%–20.4%)with known CTLs.To study the functions of these two lectins,we expressed recombinant(r)Lec1 and Lec2 in Escherichia coli and tested their ability of agglutination of red blood cells(RBCs)and interaction with microorganism from neritic and deep-sea environments.To obtain bacteria from deep-sea hydrothermal field,we isolated and cultured five deep-sea bacteria from the same deep-sea hydrothermal field as Alaysia sp.,one of these bacteria is a potential novel species,then the genetic and phenotypic analysis were used to clarify its taxonomic status.The result indicated that this strain represents a novel species in the genus Alteromonas,for which the name Alteromonas oceani sp.nov.is proposed(type strain S35~T=KCTC 52449~T=CGMCC 1.16029~T).The subsequent results show that rLec1 and rLec2 can bind a wide arrange of bacteria from neritic and deep-sea environments,including Alteromonas oceani sp.nov.,however the binding indexes of two lectins differed with different bacteria;The activities of rLec1 and r Lec2 were most stable and highest at 4°C,and decreased at higher temperatures;In addition to bacterial binding activities,r Lec1 also exhibited apparent agglutinating activities against most of the bacteria in a calcium-independent manner,whereas rLec2 could not agglutinate any of the bacteria;Both lectins inhibited bacterial growth of the Gram-positive bacteria S.iniae.That hemagglutination assay showed both rLec1 and r Lec2 caused agglutination of RBCs from rabbit,chicken,and fish(turbot)in the presence of calcium.These results provided the first insights into the functional properties of CTLs in deep-sea Alaysia sp..