The Expression And Function Analysis Of Lamprey Cathepsin D And Cysteine-rich Buccal Gland Protein PR-1 And CRD Domain

Cathepsin D(CTSD)is an aspartic proteinase which is present in the lysosome and plays important roles in the process of protein degradation.The primitive jawless lampreys usually live on the blood and flesh of host fish.The previous studies have shown that cathepsin D(Lamprey-cathepsin D,L-CTSD)is expressed in the buccal gland of lampreys.However,the functions of L-CTSD have not been reported yet.In order to study the functions of L-CTSD,pCold?-L-CTSD was constructed and successfully expressed in Escherichia coli Rosetta.SDS-PAGE showed that recombinant L-CTSD(rL-CTSD)was expressed as the inclusion bodies.The soluble protein with the molecular weight of 43 kDa was obtained and migrated as a single band after denaturation and renaturation.In addition,pcDNA3.1-L-CTSD was also successfully constructed and transfected in 293 T cells by liposome.The soluble rL-CTSD was got through affinity chromatography.The recombinant protein was identified as rL-CTSD by using Western Blot and mass spectrometry.Both expressed rL-CTSD could degrade hemoglobin,fibrinogen and serum alumin in acidic conditions,which are the main protein component of blood.This suggests that L-CTSD plays important roles in the feeding and digestion processes of lampreys.In addition,the proliferation of Escherichia coli could be inhibited by both rL-CTSD,which indicates L-CTSD participates in the immune response of lampreys.Thus,the expression and functions studies of L-CTSD will not only help to illuminate the feeding mechanism of lampreys,but also protect them from death.Furthermore,the lamprey buccal glands also contain cysteine-rich secretory protein family member: cysteine-rich buccal gland protein(CRBGP),which is composed of a pathogenesis-related group 1 domain(PR-1)and a cysteine-rich domain(CRD).Previous studies have shown that CRBGP plays important roles in the regulation ion channels and has the anti-angiogenic activity.In order to illuminate the relationship between functions of CRBGP and its PR-1 and CRD domains,pET42a-L-PR-1 and pET42a-L-CRD were successfully constructed.The recombinant L-PR-1(rL-PR-1)and L-CRD(rL-CRD)were expressed as soluble proteins with the molecular weight of 52 kDa and 39 kDa,respectively.According to the assay of MTT,both rL-PR-1 and rL-CRD could inhibit the proliferation of human umbilical vein endothelial cells.In addition,the inhibition effect induced by rL-CRD was more strong than that of rL-PR-1,suggested that CRD domain plays important roles in L-CTSD was up-regulated in the above tissues,which suggested that L-CTSD may play the inhibition of HUVEC proliferation,Thus,the expression of PR-1 and CRD domain provide the basis for further studies of the functions of CRBGP.