The Cloning, Expression And Anti-angiogenic Activity Of A Novel Cysteine-rich Secretory Protein From The Buccal Gland Of Lampetra Japonica

Cysteine-rich secretory protein (CRISP) which is widely distributed in mammalian andnon-mammalian plays important roles in sperm maturation, sperm-egg fusion, immunedefense, and ion channels regulation. In addition, it is also a good marker for the detectionand prevention of clinical diseases. Lampreys are one of the most primitive jawlessvertebrates which usually attack the host fishes for blood in the sea water. Recently, a novelcysteine-rich secretory protein family member (cysteine-rich buccal gland secretory protein,CRBGP) was separated from the buccal gland of lampreys. However, most studies havefocused on mammals and reptiles CRISP genes, and little work has been done on lampreyCRISP gene. For a long time, lampreys are used to study the phylogeny and evolution ofvertebrates because of its unique position. In the present study, the quantitative Real-TimePCR has shown that Lamprey CRBGP (L-CRBGP) was widely distributed in the heart, buccalgland, kidney, leukocytes, gill, liver, and intestines of the lampreys. After LPS stimulation,L-CRBGP is up-regulated significantly in the heart, buccal gland, and kidney of the lampreys,suggested that CRBGP may participate in the process of feeding and immune defense oflampreys. In addition, the L-CRBGP gene was constructed into pGEX-4T-1prokaryoticexpression vector which is induced by IPTG in E.coli Rosetta, and successfully expressed as asoluble fusion protein with the molecular weight of51.6kDa. And the recombinantL-CRBGP (rL-CRBGP) was purified from GST affinity chromatography. In addition, thenative CRBGP was purified from the buccal gland of lampreys by gel filtration.Both native CRBGP and rL-CRBGP have antioxidant activity through vitro experiments.In addition, both native CRBGP and rL-CRBGP could inhibit the proliferation of HUVECand Hela cells by using MTT assay. Compared with native CRBGP, the activity ofrL-CRBGP is not obvious probably due to the pGEX-4T-1expression vector on therecombinant protein which could not modify the rL-CRBGP after translation. Furthermore,native CRBGP was found to suppress the migration and invasion of HUVEC and Hela cells.And native CRBGP was also found to induce apoptosis in Hela cells. Finally, chorioallantoicmembrane (CAM) model showed that native CRBGP has anti-angiogenesis features. Tobetter elucidate the anti-angiogenic activity and mechanism of lampreys CRBGP will not onlyhelp us clarify the physiological functions of CRBGP in lampreys, but also provide muchmore information on the development of novel antiangiogenic drugs in the furture.