Molecular mechanism of the intracellular trafficking of parasite cathepsin L using GFP expression system

1998 Fiscal Year Final Research Report Summary

• Project/Area Number: 09670271
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 寄生虫学(含医用動物学)
• Research Institution: Juntendo University School of Medicine
• Principal Investigator: YAMASAKI Hiroshi Juntendo Univ.Sch.of Med., Assistant prof., 医学部, 講師 (00138207)
• Project Period (FY): 1997 – 1998
• Keywords: Fasciola sp. / cathepsin L gene / pro-peptide deletion / transfection / GFP / rat pituitary tumor cell / confocal raser scannig microscope

Research Abstract

Fasciola cathepsin L (CL) is synthesized as an inactive preprocathepsin L consisting of three domains (N-terminal pre-peptide, pro-peptide and mature enzyme). It has been known that pre-peptide functions as signal sequence to be transported into endoplasmic reticulum, but there is little information about the function of the pro-peptide. In the present study, in order to clarify the function of the pro-peptide, 4 mutated cathepsin L genes which are partially deleted pro-peptide were amplified by polymerase chain reaction (PCR) and designated as prepro*l8CL, prepro*39CL, prepro*67CL, and prepro*88C, respectively. In addition, wild type gene (preproCL), pre-peptide deleted (proCL) and entire pro-peptide deleted genes (mCL) were also amplified by PCR.These genes were inserted into pEGEP-N1 vector for protein expression. The vector contains the unique green fluorescent protein (GFP) variant gene for fusing target proteins to the N-terminus of GFP and GFP fusin proteins can be used for stud ying protein localization and trafficking. Seven different recombinant plasmids carrying a mutated cathepsin L gene were transfected into rat pituitary tumor cell (GI-14C1) using Fugene 6 reagent. The transient expression of the mutated cathepsin L fused with GUP was examined tinder fluorescence and con focal raser scanning microscopes for 961w after transfection. The GFP as a control was expressed throughout the cytosol and cell surface. When the mutated genes were transfected, however, the cathpsin Ls fused with GFP were localized at the granules in the peripheral region of the GH4Cl cells. The granules seems to be secretory granules by immunocytochemistry using anti-rat prolactin antibody. Because it has well known that the prolactin is localized in the secretory granules in the GH4Cl, and the mutated cathepsin Ls fused with GFP were also localized at same granules. As conclusion, the relationship with pro-peptide and trafficking of cathepsin L in the GH4Cl cell is still unclear and the further study would be needed to specify the localization sites of the mutated cathepsin L using another fluorescent probes.