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Biomedical subjects

S E Kane

Publications and source records attributed to S E Kane.

At least 19 recordsLinked to original sources
PubMed2001

MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell lines.

The human multidrug resistance-1 gene (MDR1) is a dominant selectable and amplifiable marker in mammalian tissue culture cells. MDR1 is also being investigated as a gene therapy tool, both to protect normal cells against chemotherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes. The success of these strategies will depend on whether MDR1 expression can be sustained at levels high enough to confer a survival advantage on target cells. However, the MDR1 selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug. The current report is a detailed molecular analysis of MDR1 selection stringency compared with the common neo selectable marker. A bicistronic vector encoding MDR1 and neo genes linked through an internal ribosome entry site was transferred into NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDR1 expression) or to G418 (to select for neo expression). Surviving populations and individual clones of cells were analyzed for expression levels of MDR1 and neo gene products; resistance to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vector DNA. These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expression can be accommodated primarily by gene amplification, even within an individual transduced clone and starting from a single-copy proviral integration event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection. Taken together, these results have important implications for the potential utility of MDR1 as a selectable marker and as a gene therapy tool in hematopoietic cells.

3T3 Cells↗
PubMed2000

Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin.

One mechanism by which chemotherapeutic agents kill tumor cells is by induction of apoptosis. Basic fibroblast growth factor (bFGF/FGF-2) has been reported to inhibit apoptosis in NIH 3T3 cells treated with chemotherapy drugs. We have investigated how bFGF modulates apoptosis induced by cisplatin in NIH 3T3 cells. Treatment with 10 microgram/ml cisplatin for 12 h induced apoptosis in 2 to 13% of the cells at 24 h post-treatment. Preincubation with 10 ng/ml bFGF for 24 h led to cisplatin-induced apoptosis in 20% to 50% of the cells. Preincubation with lower concentrations of bFGF (0.1-1 ng/ml) or simultaneous addition of bFGF and cisplatin had no effect on the amount of apoptosis. Pretreatment with bFGF also significantly decreased the dose-dependent survival of NIH 3T3 cells exposed to cisplatin, as determined by colony formation. Cells treated with 10 ng/ml bFGF showed a distinct morphology, appearing smaller and more refractile, before cisplatin exposure. The enhancement of cisplatin-induced apoptosis and the morphology shift demonstrated the same dose response to bFGF, and both effects were reversible if bFGF was removed from the medium for 24 h before cisplatin treatment. Mitogenic response to bFGF by NIH 3T3 cells saturated at 0.5 ng/ml, as measured by (3)H-thymidine uptake, and this response was blocked by coaddition of suramin, an inhibitor of FGF ligand-receptor interactions. Suramin did not reverse the enhancement of cisplatin-induced apoptosis by bFGF. Therefore, bFGF sensitized NIH 3T3 cells to cisplatin, and this effect might be mediated through a pathway separate from that used for mitogenic signaling.

3T3 Cells↗
PubMed1999

Cmdr1, a chicken P-glycoprotein, confers multidrug resistance and interacts with estradiol.

We have cloned from a chicken intestinal cDNA library Cmdr1, the first avian P-glycoprotein. Cmdr1 is 67% and 69% identical to proteins encoded by the human MDR1 and MDR2 genes, respectively. Functional expression of Cmdr1 in both mouse NIH 3T3 and yeast cells demonstrated that Cmdr1 represents the avian ortholog of human Mdr1, since it confers resistance to several anticancer drugs and the fluorescent dye rhodamine 6G. Northern and immunoblot analysis showed that CMDR1 is highly expressed throughout the intestine and in the liver, and to a considerable extent in kidney, brain, lung, heart, eye and follicles. In situ hybridization revealed a cell type-specific expression of CMDR1 in the intestinal epithelium, with high levels in the villi of the small and large intestine as well as crypt cells. These data suggest that Cmdr1 could play a role in intestinal detoxification. Most interestingly, immunoblotting showed that Cmdr1 is also expressed in ovarian tissues, particularly in theca cells, the major site for ovarian estrogen production in birds. Indeed, competition experiments indicated that Cmdr1 interacts with estradiol, since rhodamine 6G efflux was efficiently blocked by estradiol in NIH 3T3 cells expressing Cmdr1. Rhodamine efflux was also blocked by PSC-833, a specific inhibitor of steroid-transporting P-glycoproteins from mammalian cells. We propose that Cmdr1 in ovarian cells could be involved in the cell type-specific transport or release of estrogen that is essential for avian follicular development.

3T3 Cells↗
PubMed1998

Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L.

Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells. However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear. We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization. High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium. At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes. Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium. Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution. Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion. Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT L, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium. We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.

3T3 Cells↗
PubMed1998

Construction and characterization of single-transcript tricistronic retroviral vectors using two internal ribosome entry sites.

We describe a series of retroviral vectors containing two internal ribosome entry sites (IRES) for the co-transcription of three genes. Transcription of the single-transcript tricistronic mRNA is under the control of a Harvey murine sarcoma virus long terminal repeat. The 5'-most open reading frame is under either cap-dependent or cap-independent translational control, while the two downstream open reading frames are translated in a cap-independent fashion using the initiation codons of their respective IRES elements. Both IRES elements are taken from the encephalomyocarditis virus. To characterize these vectors, we used the human multidrug resistance gene (MDR1) in the 5' position, the gene for green fluorescent protein (GFP) in the middle position, and neo in the 3' position. The vectors were either transfected directly into NIH3T3 mouse fibroblasts or packaged into retrovirus and then transduced into NIH3T3 cells. Gene transfer was followed by selection with colchicine, which selects for expression of the MDR1 gene, or with G418, which selects for expression of the neo gene. Thus, we could determine the function of the tricistronic vectors under conditions of selection for either the 5'-most or the 3'-most gene. In DNA-mediated transfections, we were able to achieve expression of all three open reading frames under either selection condition. We obtained higher expression of all three genes when colchicine was used to select for MDR1 expression than when G418 was used to select for neo expression. Expression of the non-selected GFP gene (the middle cistron) was unstable, most likely due to loss of integrated GFP DNA sequences during long-term culturing. We were able to achieve retrovirus-mediated transduction of all three genes, but this was an inefficient process.

3T3 Cells↗
PubMed1998

Effect of carbohydrate position on lysosomal transport of procathepsin L.

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30-50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.

3T3 Cells↗
PubMed1998

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Previous studies have shown that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the enzyme thymidylate synthase (TS). The aim of this study was to determine whether the chemosensitivity of human cancer cells to fluoropyrimidines could be increased by decreasing TS expression with antisense oligodeoxyribonucleotides (ODNs). ODNs (18-mers) targeted at the AUG translational initiation site of TS mRNA inhibited translation in a sequence- and dose-dependent manner in a rabbit reticulocyte lysate in vitro translation system. Treatment of human colon cancer HT-29 cells with antisense ODNs decreased TS catalytic activity in the cells in a dose-dependent manner over a short period, but the longer-term effect of the TS antisense ODN treatment was actually to increase the amount of TS in the cells and to decrease their sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd). However, when human nasopharyngeal cancer KB31 cells were transfected with a plasmid (pHaMAGRP) construct containing the TS antisense fragment (+ 1 to + 422) under the control of a glucose-regulated promoter, the expression of both TS protein and TS catalytic activity was decreased by nearly 30% (P = 0.014), and sensitivity of these cells to FdUrd was enhanced by approximately 8-fold (P = 0.021). No changes in the levels of expression of TS protein or FdUrd-associated cytotoxicity were observed in control, vector-transfected cells. No change was observed in the sensitivity of transfected cells toward either cisplatin or Adriamycin. These results show that the level of expression of TS in human malignant cells can be down-regulated with antisense TS RNA, and their sensitivity to fluoropyrimidines can, thereby, be increased.

Animals↗
PubMed1997

Alteration of substrate specificity by mutations at the His61 position in predicted transmembrane domain 1 of human MDR1/P-glycoprotein.

In CFTR, a member of the ABC superfamily and a chloride channel, amino acid substitutions in its transmembrane domains 1 and 6 (TM1, TM6) have been reported to modulate the anion selectivity or ion conductance of the ion channel. In P-glycoprotein, no amino acid substitution in TM1, but some in TM6, have been reported to modify the substrate specificity of this protein. In this work, we demonstrated the involvement of His61, which is in the middle of the predicted TM1, in the function of P-glycoprotein. His61 was replaced by all other amino acid residues, and each of the mutant cDNAs was introduced into drug-sensitive human carcinoma cells, KB3-1. The drug-resistance profile of cells stably expressing each mutated P-glycoprotein was investigated by comparing their relative resistance to vinblastine, colchicine, VP16, and adriamycin. The resistance to vinblastine was increased by replacing His61 by amino acids with smaller side chains, while it was lowered by replacing by amino acids with bulkier side chains. The reverse effect was observed for resistance to colchicine and VP16. The resistance to adriamycin was increased by replacing by amino acids with bulkier side chains except Lys or Arg, which have a basic side chain. We also showed that the replacement of His61 by Phe and Lys greatly impaired the efflux of calcein AM, while the replacement had no effect on the efflux of rhodamine 123. These results suggest that an amino acid residue at position 61 in TM1 is important in deciding the substrate specificity of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Abnormal glycosylation of procathepsin L due to N-terminal point mutations correlates with failure to sort to lysosomes.

A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in glycosylation of a normally cryptic site near its C terminus. When alanine replaced His36, Arg38, or Tyr40, the nascent chain of the mutant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn268. In contrast, when alanine replaced Ser34, Arg37, or Leu39, this second carbohydrate chain was not added. This alternating pattern of abnormal glycosylation suggested that propeptide residues 36-40 normally assume an extended conformation having the side chains of residues 36, 38, and 40 facing in the same direction. When tyrosine conservatively replaced His36 or lysine replaced Arg38, Asn268 was not glycosylated. But the procathepsin L mutant having phenylalanine in place of Tyr40 was glycosylated at Asn268, which indicates that the hydrogen bond between the hydroxyl group of Tyr40 and the carboxylate group of Asp82 is necessary for normal folding of the nascent proenzyme chain. Mutation of the adjacent alpha2p (ERININ) helix of the propeptide or addition of a C-terminal epitope tag sequence to procathepsin L also induced misfolding of the proenzyme, as indicated by addition of the second oligosaccharide chain. In contrast, the propeptide mutation KAKK99-102AAAA had no effect on carbohydrate modification even though it reduced the positive charge of the proenzyme. Misfolded mutant mouse procathepsin L was not efficiently targeted to lysosomes on expression in human HeLa cells, even though it acquired phosphate on mannose residues. The majority of the mutant protein was secreted after undergoing modification with complex sugars. Similarly, epitope-tagged mouse procathepsin L was not targeted to lysosomes in homologous mouse cells but was efficiently secreted. Since production of mature endogenous protease was not reduced in cells expressing the tagged protein, the tagged protein did not compete with endogenous procathepsin L for targeting to lysosomes.

3T3 Cells↗
PubMed1997

Nucleoprotein-based nanoscale assembly.

A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M-HhaI and M x MspI. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M x HhaI or FCGG for M x MspI), we show that the first recognition sequence becomes an address for M x HhaI, while the second sequence becomes an address for M x MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M-HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.

Base Sequence↗
PubMed1996

Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene.

We describe a series of two-gene and bicistronic retroviral vectors that use the human MDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we used neo as a reporter gene for foreign gene expression and as an independently selectable marker for comparison with MDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. Using MDR1-neo virus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting that MDR1 is a more stringent selectable marker than neo in NIH 3T3 and KB-3-1 cell lines. Whereas neo gene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of the MDR1 gene product than did selection with G418, suggesting that the stringent MDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carrying MDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility for in vitro and in vivo applications of gene transfer technology, including gene therapy.

3T3 Cells↗
PubMed1995

Transduction of NIH 3T3 cells with a retrovirus carrying both human MDR1 and glutathione S-transferase pi produces broad-range multidrug resistance.

In the experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units. We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous gene along with MDR1 [M. E. Metz, D. M. Best, and S. E. Kane. Virology, 208: 634-643, 1995]. To test the ability of this vector to deliver two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase pi (GST pi, the most abundant member of the glutathione S-transferase family in human tumor cells) was inserted into pHaMASV, and this plasmid was transfected into ecotropic packaging cells. The resulting pHaMASV.GST pi ecotropic retrovirus, which was produced at a titer of 2 x 10(6) colony-forming units/ml, was used to transduce NIH 3T3 cells. After initial selection in 60 ng/ml colchicine, a population of transduced cells was exposed to stepwise increasing colchicine concentrations to select for amplified expression of MDR1. As MDR1 expression increased, the expression of GST pi increased in concert, as demonstrated by Northern analysis, Western analysis, and measurement of glutathione S-transferase activity. Transduced cells growing in 1280 ng/ml colchicine had about 3-fold higher total glutathione S-transferase activity than nontransduced cells and 2.5-fold higher activity than transduced cells growing in 60 ng/ml colchicine. Northern hybridizations demonstrated a 3-5-fold increase in both the full-length retroviral message encoding MDR1 and the subgenomic mRNA encoding GST pi after amplification of resistance from 60 to 1280 ng/ml colchicine. The cytotoxic effects of several xenobiotics were evaluated in NIH 3T3 cells transfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST pi retrovirus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expressed. 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of MDR1 had identical levels of resistance to doxorubicin or colchicine. These results suggest that GST pi expression did not contribute to doxorubicin resistance in this model system. However, 3T3.GST640 cells were about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrobenzene compared to cells expressing MDR1 alone, consistent with the ability of GST pi to conjugate both of these cytotoxins. Increases in drug resistance paralleled increases in gene-specific mRNA and recombinant protein levels in all cases.4+ chemotherapy.

3T3 Cells↗
PubMed1995

Harvey murine sarcoma virus/MDR1 retroviral vectors: efficient virus production and foreign gene transduction using MDR1 as a selectable marker.

Retroviruses are used for a variety of applications requiring the delivery of exogenous genes to cells and animals. For many of these applications, including gene therapy, safer and more efficient retroviral vectors are needed. Vectors based on Harvey murine sarcoma virus (HaMSV) are attractive because nearly all their viral sequences outside of the LTRs are derived from rat endogenous VL30 retroviruses. These sequences are not homologous to the functional viral mRNAs in commonly used retrovirus packaging cell lines, the packaging and dimerization domains of HaMSV are small and contain no splice donor sites, and the 5' sequences of HaMSV appear to confer efficient packaging and stability on genomic RNAs. HaMSV/MDR1 vectors use the human multidrug resistance gene as a dominant, selectable, amplifiable marker for gene delivery, but current versions of these vectors are large, with over 3300 nt of HaMSV sequences downstream of MDR1. We analyzed the requirement for these downstream sequences in HaMSV vectors and found that modified HaMSV/MDR1 vectors lacking virtually all viral sequences downstream of MDR1 support the production of high-titer retroviruses and the efficient transduction, selection, and amplification of MDR1. A reduced-size HaMSV/MDR1 vector was further modified to include a second heterologous gene under the control of an internal SV40 promoter. Using MDR1 as a selectable marker, we obtained efficient virus production, gene transduction, and expression of MDR1 plus the heterologous gene.

3T3 Cells↗
PubMed1995

Use of the stress-inducible grp78/BiP promoter in targeting high level gene expression in fibrosarcoma in vivo.

Current advances in human gene therapy open up new frontiers for molecular therapies of cancer. However, one major limitation in cancer gene therapy is the lack of a general tumor-specific promoter which allows stringent and high level expression of the therapeutic reagent in malignantly transformed but not normal tissues. Hallmark features of solid tumors such as glucose deprivation, chronic anoxia, and acidic pH induce the glucose-regulated proteins, in particular, GRP78/BiP, a M(r) 78,000 endoplasmic reticulum-localized protein with chaperone and calcium-binding properties. We report here that a truncated rat grp78 promoter with most of the distal basal elements removed can be utilized as a potent internal promoter in a retroviral vector to drive high level expression of a reporter gene in a murine fibrosarcoma model system. The stress-inducible grp78 promoter offers a novel approach for gene delivery systems targeting transcription in tumorigenic cells.

3T3 Cells↗
PubMed1993

Targeted disruption of the mouse mdr1b gene reveals that steroid hormones enhance mdr gene expression.

To evaluate the role of P-glycoprotein in steroid secretion in adrenal cells, we have used gene targeting to introduce a null mutation into one allele of the mdr1b gene in mouse Y1 adrenal cells. Characterization of both the wild-type and the mutant cell lines revealed the following. 1) The expression of mdr1b is enhanced by steroid hormones, in a feedback regulatory mechanism. Inhibition of steroid biosynthesis by 2-aminoglutethimide blocks the adrenocorticotropin (ACTH)-induced increase in mdr1b mRNA levels. 2) ACTH-stimulated steroid secretion is markedly decreased in the mutant cell line. This decreased steroid secretion in the mutant cells occurs despite an increase in the levels of mdr1b mRNA and P-glycoprotein. Kinetic analyses of vinblastine and daunomycin accumulation in both the wild-type and the mutant cell lines during ACTH-stimulated steroidogenesis show that in the mutant cells both drugs accumulated to higher levels than in Y1 cells, suggesting that the remaining mdr1b allele in the mutant cells is relatively inactive as an exporter of steroids, or that the targeted disruption of the mdr1b allele is associated with other changes in the mutant cells which block ACTH-stimulated steroid secretion.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Mouse procathepsin L lacking a functional glycosylation site is properly folded, stable, and secreted by NIH 3T3 cells.

The role of glycosylation in the synthesis, transport, and localization of procathepsin L has been analyzed. Procathepsin L is the precursor form of a lysosomal cysteine protease which is synthesized in large amounts by malignantly transformed cells and cells treated with growth factors or tumor promoters. As for other lysosomal hydrolases, procathepsin L is predominantly secreted when it is overexpressed, despite the presence of the lysosomal sorting marker mannose 6-phosphate. To study the role of glycosylation in procathepsin L trafficking, we constructed a cDNA in which the codon for Asn-204 was mutated to encode Gln. When this mutated cDNA was transfected into NIH 3T3 cells, a completely nonglycosylated form of procathepsin L was expressed. The protein appeared to be folded properly, as judged by its ability to be proteolytically processed to a lower molecular weight form during incubation at pH 4. Nonglycosylated protein was stable, both as unprocessed proenzyme and after processing at pH 4, and it was predominantly secreted instead of being delivered to lysosomes. Nonglycosylated and endogenous glycosylated procathepsin L were secreted by NIH 3T3 cells with identical kinetics. These studies therefore confirm that Asn-204 is the normal functional carbohydrate acceptor in procathepsin L and that carbohydrate in wild-type procathepsin L serves predominantly as a lysosomal targeting signal, with little or no role in protein folding or stability. Furthermore, there does not appear to be a major mannose 6-phosphate-independent route of lysosomal localization in NIH 3T3 cells. In experiments with a second mutant, the glycosylation signal at Asn-251, which normally is not utilized, was capable of serving as a carbohydrate acceptor, suggesting that there is normally a structural impediment to glycosylation at Asn-251 in procathepsin L.

3T3 Cells↗