Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.


a b s t r a c t
The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from nontransfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed. &

Value of the data
This article provides information on the construction of a vector coding for human cathepsin L-EGFP chimeras, and it describes the insert's sequence.
The data provide detailed information on the molecular forms of cathepsin L chimeras with enhanced green fluorescent protein that are expressed in the human colorectal carcinoma HCT116 cell line.
The article provides data on the effects of cathepsin L-EGFP expression in HCT116 cells with regard to proliferation rates and metabolic activity.
Data EGFP-tagging is used to visualize transport and trafficking of cellular proteins including cysteine cathepsin proteases [2]. We have shown that EGFP-tagging does not interfere with intrinsic sorting signals at the N-terminus of cysteine cathepsin proteases when the tag is attached to the C-terminus via a spacer peptide [2][3][4]. In addition, transport to endo-lysosomal compartments and other cellular destinations is not affected by the EGFP tag [2][3][4]. Likewise, proteolytic activity is pertained in cathepsin-EGFP chimeras expressed in mammalian cell lines [2][3][4][5][6][7].
We have shown that cathepsin L is sorted and mis-trafficked to the nucleus of HCT116 cells as fulllength protein and in proteolytically active form [1]. More specifically, nuclear cathepsin L accelerates cell cycle progression of HCT116 cells which are hyper-proliferative colorectal carcinoma cells. In this article, we demonstrate via expression of cathepsin L-EGFP chimeras the effects of enhanced protease expression promoting proliferation of HCT116 cells.

Experimental design, materials and methods
In this article, we describe the construction of a vector encoding human cathepsin L-EGFP (hCL-EGFP) with cDNA derived from cathepsin L mRNA of human HaCaT keratinocytes upon insertion into the multiple cloning site of pEGFP-N1 by NheI and BamHI restriction. Here, we share the relevant sequence data of phCL-EGFP. The vector is introduced through lipofection to express chimeric, EGFP-tagged cathepsin L in HCT116 cells. This colorectal carcinoma cell line was further analyzed with regard to proliferation and metabolic activity by MTT-assays upon transfection with phCL-EGFP in comparison to non-transfected and pEGFP-N1-transfected cells. The molecular forms of protein chimeras expressed in HCT116 cells were identified by immunoblotting of whole cell lysates separated by SDS-PAGE.

Vector construction
Total RNA at a concentration of 0.05 mg/mL was reverse transcribed with a cDNA cycle kit (Invitrogen, Karlsruhe, Germany) using oligo dT primers and AMV reverse transcriptase. Amplification of the complete cathepsin L coding sequence was by PCR with 500 ng cDNA using 0.5 mM each of the primers "CL for NheI" (5 0 -aca cag gtt tta aaa cat gaa tcc tac a-3 0 ) and "CL rev BamHI" (5 0 -agc tac ccc act gtg tga gct ggt gga-3 0 ), 0.125 mM each of dNTPs, and 0.4 U Phusion DNA polymerase (Finnzymes, Espoo, Finland). By this cloning strategy, restriction sites for NheI and BamHI were introduced and the stop codon was removed, thus eventually yielding the sequence for a chimeric protein with the fulllength cathepsin L sequence covalently connected to the EGFP tag by a spacer peptide linker. The final PCR product and pEGFP-N1 plasmid were each digested with NheI and BamHI (both, MBI, St. Leon-Rot, Germany). The restricted cDNA was ligated into the linearized vector using T4-DNA ligase (MBI) in the presence of ATP-containing reaction buffer (MBI) at 4°C overnight, followed by ligase inactivation at 65°C for 10 min. The ligated plasmids were transformed into competent E. coli JM 109, and kanamycin-resistant clones were used for isolation of vector cDNA (Qiagen, Hilden, Germany), which was eluted and stored in 10 mM Tris-HCl at pH 8.0. Control digests of the isolated plasmids were performed with NheI and BamHI, each used at 0.25 U/mg plasmid DNA, for insert restriction, and additionally EcoRI with a restriction within the insert was used (all, MBI) before analyzing on 1% agarose gels by electrophoresis. Finally, sequencing using the primers "CMV-Profor" (5 0 -aaa tgg gcg gta ggc gtg-3 0 ) and "EGFP-Nrev" (5 0 -cgt cgc cgt cca gct c-3 0 ) confirmed the correct product (Sequiserve, Vaterstetten, Germany).

MTT assays
Cell proliferation rates and metabolic activity levels of transfected HCT116 cells were examined by a colorimetric assay in which the yellow 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is reduced to form purple formazan crystals by intracellular NAD(P)H-oxidoreductase. The assays were performed in duplicates and repeated at least twice as previously described [8], and the reaction product was quantified at 595 nm with a Tecan GENios Reader (Tecan Deutschland GmbH, Crailsheim, Germany).

Results
Cathepsin L has been shown to be mis-trafficked in HCT116 cells and to enter the nucleus as an unexpected scene of potential action [1]. Nuclear cathepsin L is suggested to involve in regulation of cell cycle progression of HCT116 cells by accelerating S phase [1]. Moreover, the vector phCL-EGFP coding for EGFP-tagged cathepsin L was expressed in HCT116 cells and sorted to the nucleus [1, Fig. 7D 0 ].

Construction of the phCL-EGFP vector
Vector construction was performed upon cloning of human cathepsin L from non-tumorigenic HaCaT keratinocytes and cDNA was inserted into the pEGFP-N1 vector (Clontech, Heidelberg, Germany), resulting in the fusion of sequences coding for cathepsin L and eGFP in phCL-EGFP. The cloning strategy (Fig. 1) involved removal of the stop-codon at the 3 0 -end of cathepsin L cDNA, and introduced restriction sites for NheI and BamHI at the 5 0 -and 3 0 -ends, respectively, to ensure that the complete coding sequence was amplified. Removal of the stop-codon by BamHI digestion permitted further transcription once the gene was ligated in-frame into the suitable vector pEGFP-N1 to obtain mRNA coding for a fusion construct of cathepsin L and the marker protein enhanced GFP.
Transformed E. coli colonies with the cathepsin L sequence inserted into the EGFP-encoding vector were cultivated in kanamycin-containing LB medium, and plasmids were harvested from this broth of Fig. 1. Scheme of specific amplification primers and expected amplicon. Forward and reverse primers for gene-specific amplification of cathepsin L are indicated in red and their amplicon is highlighted in green. Cleavage sites of NheI and BamHI are indicated by arrows. Numbers denote the length of the gene. resistant bacteria. As an additional control of proper vector construction, the purified plasmids were digested with NheI and BamHI to gain the insert, and additionally with EcoRI, which cleaves once within the cathepsin L sequence. Therefore, after digest of cathepsin L-EGFP-containing plasmids, fragments with a length of 4700 bp, 760 bp, and 259 bp were expected. The preparations were analyzed by agarose gel electrophoresis, and three bands of the expected size were observed in the digests of phCL-EGFP (Fig. 2).
Next, the plasmid phCL-EGFP was sequenced from downstream (starting 5 0 ) and upstream (starting 3 0 ) in the vector (Fig. 3). Alignment of this cDNA sequence with the reported human cathepsin L sequence (preprocathepsin L precursor, Homo sapiens, accession number GenBank AAA66974.1) revealed 100% identity, confirming that cathepsin L-coding cDNA was inserted into the pEGFP-N1 vector as shown in the vector map of phCL-EGFP (Fig. 4).   3. Partial sequence of phCL-EGFP. Nucleotide sequence of insert region of phCL-EGFP as revealed by up-and down-stream sequencing reactions is depicted. The cathepsin L-coding sequence is highlighted in light green, sequences of the surrounding vector are labeled in red, start-codons ATG of cathepsin L and EGFP, respectively, are underlined, primers are marked in dark green, the cleavage site of NheI is shown in black, the restriction site of BamHI is marked in pink, and the EcoRI site in grey, while the spacer sequence between cathepsin L and EGFP is color-coded in dark blue.

Transfection of HCT116 cells with phCL-EGFP
Sub-confluent cultures of HCT116 cells were transiently transfected with phCL-EGFP yielding expression of full-length cathepsin L, which was co-localized with endogenous cathepsin L and also reached the nuclei (see Fig. 7D 0 in [1]). In addition, MTT assays were performed with non-transfected, pEGFP-N1-, and phCL-EGFP-transfected HCT116 cells after 24 h. Data revealed that proliferation rates were not altered upon expression of enhanced GFP alone, while the MTT conversion rates were increased in HCT116 cells expressing hCL-EGFP chimeras (Fig. 5), although this increase did not reach significance over non-transfected controls.

Molecular forms of cathepsin L-EGFP chimeras translated in HCT116 cells
To identify the molecular forms of the cathepsin L-EGFP chimeras translated in HCT116 cells, immunoblotting was performed with whole cell Iysates of non-transfected cultures and with those expressing pEGFP-N1 or phCL-EGFP, respectively. The expected molecular forms of cathepsin L were Fig. 4. Vector map of phCL-EGFP. Scheme of the 5.7-kb-vector phCL-EGFP coding for full-length cathepsin L-EGFP chimeric protein is depicted. Note that cathepsin L was cloned into the multiple cloning site (MCS) of the pEGFP-N1 vector (Clontech) using NheI and BamHI as indicated. Additionally the restriction site for EcoRI within the cDNA sequence that was used for control digests is marked (arrow). Numbers denote the size of full-length cathepsin L-coding cDNA and that of the expected fragments after control digest. detected in all samples, i.e. the pro-form as well as the single chain (SC) and heavy chain (HC) of the two-chain form of the protease. Lysates of phCL-EGFP-transfected HCT116 cells featured an additional band at 65 kDa that was recognized by cathepsin L-specific antibodies (Fig. 6), and which was absent from non-transfected cells or those transfected with the empty vector pEGFP-N1. The data indicates that the band at 65 kDa is representative of the hCL-EGFP chimeric protein which was stable for 24 h in HCT116 cells because no degradation bands could be observed in immunoblots with cathepsin Lspecific antibodies (Fig. 6).