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Abstract
Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1α/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1α system in embryonic kidney cells.
| Abbreviations | ||
| Ala-pNA | = | alanine-p-nitroanilide |
| APN | = | aminopeptidase N |
| DPIV | = | dipeptidyl peptidase IV |
| ECL | = | enhanced chemiluminescence |
| EGFP | = | enhanced green fluorescence protein |
| FACS | = | fluorescence activated cell sorter |
| FBS | = | fetal bovine serum |
| G-CSF | = | granulocyte colony stimulating factor |
| HRP | = | horseradish peroxidase |
| JAK/STAT3 | = | Janus kinase/signal tranducer and activator of transcription 3 |
| MAPK | = | mitogen-activated protein kinases |
| HPRT1 | = | hypoxanthine guanine phosphoribosyl transferase 1 |
| mAb | = | monoclonal antibody |
| MFI | = | mean specific fluorescence intensity |
| PAGE | = | polyacrylamide gel electrophoresis |
| PBS | = | phosphate-buffered saline |
| RT-PCR | = | reverse transcriptase-polymerase chain reaction |
| PE | = | phycoerythrin |
| SDF-1 | = | stromal cell-derived factor-1 |
| SDS | = | sodium dodecyl sulfate |
| wt | = | wild type |
| Abbreviations | ||
| Ala-pNA | = | alanine-p-nitroanilide |
| APN | = | aminopeptidase N |
| DPIV | = | dipeptidyl peptidase IV |
| ECL | = | enhanced chemiluminescence |
| EGFP | = | enhanced green fluorescence protein |
| FACS | = | fluorescence activated cell sorter |
| FBS | = | fetal bovine serum |
| G-CSF | = | granulocyte colony stimulating factor |
| HRP | = | horseradish peroxidase |
| JAK/STAT3 | = | Janus kinase/signal tranducer and activator of transcription 3 |
| MAPK | = | mitogen-activated protein kinases |
| HPRT1 | = | hypoxanthine guanine phosphoribosyl transferase 1 |
| mAb | = | monoclonal antibody |
| MFI | = | mean specific fluorescence intensity |
| PAGE | = | polyacrylamide gel electrophoresis |
| PBS | = | phosphate-buffered saline |
| RT-PCR | = | reverse transcriptase-polymerase chain reaction |
| PE | = | phycoerythrin |
| SDF-1 | = | stromal cell-derived factor-1 |
| SDS | = | sodium dodecyl sulfate |
| wt | = | wild type |