Karin Frank

Extracellular cysteines define ectopeptidase (APN, CD13) expression and function.

Abstract Alanyl aminopeptidase (APN) is a surface-bound metallopeptidase that processes the N-terminals of biologically active peptides such as enkephalins, angiotensins, neurokinins, and cytokines. It exerts profound activity on vital processes such as immune response, cellular growth, and blood pressure control. Inhibition of either APN gene expression or its enzymatic activity severely affects leukocyte growth and function. We show here that oxidoreductase-mediated modulations of the cell surface thiol status affect the enzymatic activity of APN. Additional evidence for the pivotal role of extracellular cysteines in the APN molecule was obtained when substitution of any of these six cysteines caused complete loss of surface expression and enzymatic activity. In contrast, the transmembrane Cys24 appears to have no similar function. Enzymatically inactive cysteine mutants were retained in the endoplasmic reticulum as shown by high-resolution imaging and Endoglycosidase H digestion. In the absence of any crystal-structure data, the demonstration that individual extracellular cysteines contribute to APN expression and function appears to be of particular importance. The data are the first to show thiol-dependent modulation of the activity of a typical surface-bound peptidase at the cell surface, probably reflecting a general regulating mechanism. This may relate to various disease processes such as inflammation or malignant transformation.

Quantification of aminopeptidase N mRNA in T cells by competitive PCR

The aminopeptidase N (CD13, EC 3.4.11.2) is a well‐characterized surface molecule expressed in a variety of cell types and species. Recent data indicate an expression of the APN mRNA and the corresponding aminopeptidase activity in human peripheral T cells and related cell lines as well. Here, the sensitive method of competitive PCR was used to quantify low amounts of APN mRNA in T cell lines. An APN cDNA fragment en‐shortened by a deletion of 87 bp was used as an internal APN‐specific standard. The myelo‐monocytic cell line U937 and the lymphoid T cell lines HuT78 and H9 contain 2.3 × 107, 5.9 × 106 and 5.6 × 106 copies/μg total RNA, corresponding to 160, 70 and 50 copies/cell, respectively. These data have been confirmed by determination of the APN activity, that represents a fraction only of the total cellular neutral aminopeptidase activity in hematopoetic cells. In the case of the CD13‐positive cell line U937, ∼60–70% of the total neutral aminopeptidase activity could be attributed to APN. In contrast, only a minor fraction (5–20%) of the cellular neutral aminopeptidase activity in the T cell lines H9 and HuT78 represents APN. The results suggest that APN gene expression within the hematopoetic system is not restricted to myelo‐monocytic cells, instead a low APN expression may be a common feature of lymphocytes, at least of T cells, too.

Rapid mitogen-induced aminopeptidase N surface expression in human T cells is dominated by mechanisms independent of de novo protein biosynthesis

Abstract The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.

Antisense-Mediated Inhibition of Aminopeptidase N (CD13) Markedly Decreases Growth Rates of Hematopoietic Tumour Cells

There are several reports describing neutral aminopeptidase activities expressed in immune cells1–7. One of the best studied members of these enzymes is the Zn-dependent aminopeptidase N (CD13, E.C.3.4.11.2, APN) Within the hematopoietic system the expression of this leukocyte surface antigen was thought to be restricted to myelomonocytic cells 1,8,9. During recent years, it has been proven that lymphoid cells contain APN-mRNA and express corresponding enzymatic activity, too 2–4,7,10,11. These cells contain very low amounts of CD13 only, and therefore, are predominantly CD13-negative1,9,12,13. Recently, different groups have been provided conclusive evidence that CD13 is strongly induced during processes such as T cell activation or inflammation, suggesting that this antigen may well be involved in the regulation of proliferation and differentiation processes3,12,14–15.

The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up‐regulated during mitogenic stimulation of peripheral T‐cells. In this study, we demonstrate that the half‐life of APN mRNA was considerably prolonged in these cells leading to a 2.7‐fold increase of APN transcript level. The apparent half‐life time of the APN transcript was investigated by the RNA synthesis inhibitor‐chase method using actinomycin D. The steady‐state APN mRNA levels was determined by a competitive RT‐PCR. The half‐lives estimated in resting T‐cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run‐on assays revealed that the APN gene expression of stimulated T‐cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post‐transcriptional level in addition to promoter‐mediated regulation.

ACTIVATION-DEPENDENT INDUCTION OF T CELL ALANYL AMINOPEPTIDASE AND ITS POSSIBLE INVOLVEMENT IN T CELL GROWTH

Membrane alanyl aminopeptidase (APN, EC 3.4.1 1.2) is a 150-kDa metalloprotease which has been identified as the leukocyte surface differentiation antigen CD131. In humans the APN gene is located on the long arm of chromosome 15 (ql 1-gter)2 with the coding part of the gene encoded by 20 exons3. Within the haematopoetic system APN is dominantely expressed in cells of the myelo-monocytic lineage and is used, therefore, as a standard marker in the diagnosis of leukaemia. Aminopeptidase N of leukocytes is supposed to be involved in the degradation of neuropeptides4–7 and cytokines8,9, but its function remains to be fully elucidated. APN may function as a corona virus receptor10–13 and seems to contribute in tumor invasion and matrix degradation14–15. APN is also implicated in antigen processing16. Furthermore, anti-CD13 monoclonal antibodies have been shown to neutralize CMV17. In recent years evidence accumulated showing that malignant B and T cells18–23 as well as activated T cells24–26 are capable of expressing APN on the cell surface. A similar mechanism of induction may underlie the CD13 surface expression of tumor infiltrating T cells27, or T cells28 and NK cells29 derived from local sites of inflammation.

R-esp1, a rat homologue of Drosophila Groucho, is differentially expressed after optic nerve crush and mediates NGF-induced survival of PC12 cells

The differential display reverse transcription polymerase chain reaction method was used to detect alterations in gene expression in the superior colliculus after optic nerve crush in adult rats. One of the most prominent changes observed was the selective induction of R‐esp1, a homologue of the Drosophila enhancer of split locus (Groucho). Therefore, we studied the influence of R‐esp1 on nerve growth factor (NGF)‐induced cell survival of PC12 cells. Overexpression of R‐esp1 promotes cell survival even in the absence of NGF and, conversely, it is reduced by antisense‐mediated inhibition of R‐esp1 expression. In conclusion, we propose a novel model in which R‐esp1 protein mediates the NGF‐signaling pathway.