Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein.

A protein modification essential for the cellular sorting of many biologically relevant proteins is the covalent attachment of prenyl lipids by specific transferases. Isoprenylation is known to render protein domains hydrophobic, thereby facilitating the interaction with lipid bilayers and/or membrane proteins. The target for the modification with farnesyl groups is the COOH-terminal sequence CaaX. Among the variety of farnesylated proteins the only one reported so far to be located to peroxisomes is the 37-kDa peroxisomal farnesylated hamster protein PxF. Recently we published data on the cDNA of the human gene HK33 (A. Braun et al., 1994, Gene 146: 291-295), which was revealed to be the human ortholog of PxF and was consequently renamed HsPXF. The genomic structure, molecular characterization, and evolutionary conservation of HsPXF are described herein. The exact location of the gene was defined as chromosome 1q22. The gene spans a region of approximately 9 kb, containing eight exons and seven introns. The 5' upstream region showed two potential Sp1-binding sites and an Alu repetitive sequence. Luciferase reporter activating capacity confirmed the presumed promoter activity of this region. On the transcriptional level, we detected four splice variants originating either from exon skipping or from alternative splicing events. For the HsPXF protein, a carboxyterminal farnesylation at cysteine residues was demonstrated. Through the use of HsPXF-specific antibodies, the protein was shown to be attached to the outer surface of peroxisomes. This localization together with the similarity to a peroxisomal assembly protein from Saccharomyces cerevisiae suggests HsPXF is involved in the process of peroxisomal biogenesis or assembly.

Ecto-5'-nucleotidase activity is not required for T cell activation through CD73.

It had previously been shown that CD73 (ecto-5'-nucleotidase) expressed on 30% of normal human peripheral T-lymphocytes can mediate costimulatory signals in T cell activation. To check for a possible contribution of the catalytic property of CD73 to this function transfected cell lines were created. Using site-directed mutagenesis on human CD73 cDNA the codons of His92 and His194 were exchanged for alanine codons. Wild type and mutant cDNAs were cloned into an expression vector and stably expressed in the T cell line Jurkat. Surface expression as quantitated by immunofluorescence with anti CD73 mAbs was comparable in wild type and mutant transfectant clones, whereas in contrast to the wild type both His to Ala mutants completely lacked enzymatic activity. The Jurkat transfectants were stimulated with a combination of soluble anti-CD73 mAb and PMA and their response was quantitated by IL-2 production. The responses of different clones of wild type and mutant transfectants varied within a wide range; however, the ranges of wild type and mutants overlap. It is concluded that in order to transmit costimulatory signals CD73 does not require its catalytic activity.

The antibody MY4 recognizes CD14 on porcine monocytes and macrophages.

Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74-22-15. Two-colour immunofluorescence reveals that My4 and 74-22-15 antigens are coexpressed on the same cells, and cell sorter-purified My4+ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4+ monocytes amongst all leucocytes is 5.8% with 580 cells/microliters. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four-fold lower level on AM. Treatment of cells with phosphatidylinositol-specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74-22-15. Immunoprecipitation with the My4 antibody from surface labelled pig mononuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide- (LPS)-induced interleukin-6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.

Prognostic implication of ecto-5'-nucleotidase activity in acute lymphoblastic leukemia.

Ecto-5'-nucleotidase (5'-N) activity was determined in 191 patients (71 children and 120 adults) with acute leukemia. Elevated values for 5'-N were registered in common acute lymphoblastic leukemia (ALL), but blast cells of T-cell ALL (T-ALL) and common ALL antigen-negative non-T-ALL had low enzyme activity comparable with the values of acute non-lymphocytic leukemia. Dependence of remission duration on 5'-N activity was analyzed in 74 adults with ALL, treated similarly in a prospective multicenter trial. The remission curves for ALL patients with 5'-N activity lower than 10 nmol/h x 10(6) cells were substantially and significantly better than those of patients with high activity (greater than 10 nmol/h x 10(6) cells). This difference was also evident in the immunologic subclass common ALL. Statistical evaluation showed that an interaction between immunologic subtype of the blast cells and their 5'-N activity had prognostic significance for remission duration. In addition to the independent factor, initial age, this interaction was also prognostic for survival.

Glycosylation and processing of carbohydrate side chains of ecto-5'-nucleotidase in cultured human chorionic cells.

Glycosylation and carbohydrate processing of ecto-5'-nucleotidase were studied in cultured human chorionic cells using metabolic labelling and immunoprecipitation with monoclonal antibodies. Tunicamycin blocks glycosylation altogether leading to a reduction in molecular mass of 9,500 Da. The same result is obtained by digesting the mature 72,000-Da protein with endoglycosidase F. Using various inhibitors of the carbohydrate-trimming reactions like deoxynojirimycin, deoxymannojirimycin and swainsonine smaller molecular mass reductions are observed and the oligosaccharide side chains are kept in a configuration sensitive to endoglycosidase H digestion. Digestion of mature 5'-nucleotidase with endoglycosidase H leads to a much smaller (2,000 Da) reduction in molecular mass. It is calculated that, in addition to the phosphatidylinositol-glycan anchor structure, ecto-5'-nucleotidase of human chorionic cells should carry 4 oligosaccharide side chains per subunit, 3 of which should be of the complex and one of the high mannose type. Interference with carbohydrate processing by various inhibitors does not seem to influence the distribution of ecto-5'-nucleotidase between the cell surface and intracellular membranes nor does it block the transfer of the enzyme to the phosphatidylinositol glycan anchor.

Normal ranges of the activities of ten different enzymes in 100 independent preparations of chorionic villi. Comparison of specimens from induced abortions, biopsies, and cultured cells.

In order to obtain a large set of normal control values, the activities of three cytosolic enzymes of purine metabolism and seven lysosomal enzymes were determined in homogenates of chorionic villi derived from induced abortions of normal pregnancies (7th-12th week) in about 100 individual cases. Possible reasons for the rather wide ranges of normal distributions of enzyme activities are discussed. The values are compared: (1) with available data in the literature; (2) with activities determined in decidual homogenates prepared from the same samples; (3) with activities of cells of cultures established and grown from villi in the same samples; and (4) with enzyme activities measured in chorionic biopsies using the same methods. Implications for the prenatal diagnosis of the associated metabolic diseases are considered.

Human placental ecto-5'-nucleotidase: isoforms and chemical crosslinking products of the membrane-bound and isolated enzyme.

Human placental ecto-5'-nucleotidase is specifically detected on Western blots using poly- or monoclonal antibodies. In two-dimensional electrophoresis 5'-nucleotidase, purified as well as membrane bound, is resolved in up to 13 isoforms distinguished by a different content of neuraminic acid. These forms span a range of molecular masses of about 67-71 kDa and of isoelectric points of 5.8-7.0. Chemical cross-linking of the purified enzyme with either homoor heterobifunctional reagents yields a dimer of about 140 kDa exclusively. On the other hand treatment of plasma membranes with the same reagents results in a crosslinking product of 5'-nucleotidase of about 97 kDa. The partner of the enzyme subunit in this crosslink, a protein of about 30 kDa, is unknown. Using specific antibodies the cytoskeletal components actin and tropomyosin were excluded as possible candidates.

Blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia (CML). Treatment results of 69 patients.

We have studied the clinical courses of 69 patients with blastic crises of Philadelphia chromosome positive CML to identify parameters that were associated with an increased response rate or survival. Cytogenetic analysis at the time of blastic transformation revealed additional chromosome changes in 70% of the patients tested. Bone marrow fibrosis was detected in 58% of evaluable patients. Lymphoblastic transformation was seen in 28% of the patients tested with cell surface marker analysis. The value of 5'-nucleotidase as a marker for distinguishing lymphoid from non-lymphoid blast crisis was confirmed. Of 57 evaluable patients, 23 (40%) responded to therapy (CR/PR longer than 14 days). Median survival was 75 days. Longer survival was related to the following factors: Ph1-chromosome as the only detectable cytogenetic abnormality; lymphoblastic transformation; no bone marrow fibrosis; high percentage of blasts and promyelocytes in the bone marrow, and response to therapy. No prognostic significance was associated with age, sex, Tdt, LDH, spleen size, duration of the chronic phase of the disease, white blood cell count, Hb, platelet count and percentages of basophils, eosinophils, erythroblasts and blasts and promyelocytes in the peripheral blood. These data confirm the poor prognosis of patients with blastic crisis of CML treated by conventional chemotherapy.

Qualitative and quantitative differences between Burkitt lymphoma and lymphoblastoid cell lines in the expression of membrane ecto-nucleotidases and in the response to agonists of the A2-type adenosine receptor.

The activity of ecto-nucleotidases has been determined on intact cells from Burkitt lymphoma (BL) and lymphoblastoid cell lines established in vitro (LCLs). BL cells never express ecto-5'-nucleotidase (5'-N) and exhibit only low levels of ecto-ATPase activity, whereas LCL-cells usually express both enzymes to varying degrees. There is a certain correlation (r = 0.75) between 5'-N and ecto-ATPase. When cAMP formation in response to agonists of the A2-type (stimulating) adenosine receptor is measured in the same cell lines there is no correlation with the expression of ecto-nucleotidases. Within pairs of BL and LCL cell lines derived from the same donor, an inverse relationship between ecto-nucleotidases and the response to the adenosine receptor agonist N-ethyl-carboxamido-adenosine (NECA) is observed. BL cells show a good response to NECA, whereas this is low or absent in LCL cells. Treatment of cells which exhibit both 5'-N and the adenosine receptor with specific polyclonal or monoclonal antibodies against the enzyme does not impair the function of the receptor. Antisera against peptides of the membrane antigen BNLF I-MA, coded by the EBV-genome, do not co-precipitate 5'-N out of detergent extracts of LCL-cells. In both cases 5'-N cannot be closely associated with other membrane components. The differences between BL and LCL cells in ectonucleotidases and the adenosine receptor appear to be fairly stable phenotypical markers, independent of other parameters and suitable for use in distinguishing these cells in all populations.

Is ecto-5'-nucleotidase essential for stimulation of human lymphocytes? Evidence against a role of the enzyme as mitogenic lectin receptor.

We studied with specific polyclonal and monoclonal antibodies against human ecto-5'-nucleotidase whether the enzyme, located on the surface of human peripheral lymphocytes, could function as a mitogenic receptor for the lectins PHA, Con A and PWM. Strong, but unspecific inhibitory effects on lymphocyte stimulation are observed with unfractionated antisera and ascitic fluids. However, when purified IgG from these sources is used, no such effects are found, while at the same time, complete inhibition of ecto-5'-nucleotidase activity is maintained. It is concluded that the enzyme does not act as a mitogenic receptor for the lectins. When purine de novo synthesis of the lymphocytes is blocked by aminopterin and purine nucleotides in the extracellular medium are given as the only purine source, lymphocyte stimulation becomes dependent on the enzymatic activity of ecto-5'-nucleotidase. This is independent of the lectin used. Under these conditions, enzyme activity on the 20-30% 5'-nucleotidase-positive cells is necessary and is sufficient to support the stimulation of the whole culture. In these cultures, anti-5'-nucleotidase-IgG completely depresses cell proliferation, showing clearly that this is the only enzyme on the lymphocyte surface that is capable of degrading extracellular nucleotides.

Inherited disorders of purine metabolism--underlying molecular mechanisms.

An overview of inherited disorders of purine metabolism, concentrating on well established enzyme defects is given. Included are HPRT and the LNS, APRT and 2,8-dihydroxyadenine lithiasis, hyperactivity of PRPP synthetase, ADA and PNP and immunodeficiencies. Emphasis is put on underlying molecular mechanisms on the gene-, enzyme-, or metabolite level for a better understanding of the events leading from the genotype to the clinical phenotype. Finally some aspects of extracellular purine nucleotide metabolism catalyzed by cell surface-bound ectoenzymes are discussed.

Development and properties of a monoclonal antibody specific for human ecto-5'-nucleotidase.

After immunization with purified human placental ecto-5'-nucleotidase and fusion, 18 different hybrids were obtained which produce an antibody inhibitory for enzyme activity. These antibodies show complete cross-reactivity with the enzyme in a membrane-bound form or on the surface of intact cells. After cloning and ascites production, the antibody of one clone ( IFH - 5N1 ) was studied in greater detail. The IFH - 5N1 antibody is of the IgG 1 subclass. Optimal enzyme inhibition is 90% for purified 5'-nucleotidase and 80% for the enzyme on lymphoblasts. The specificity of this antibody is further demonstrated by enzyme inhibition assays and fluorescence labeling using various 5'-nucleotidase-positive and -negative human cells such as peripheral blood lymphocytes, leukemic cells, lymphoblastoid B- and T-cell-lines and fibroblasts. The antibody should provide a useful tool for the diagnosis of certain forms of acute leukemias and for the study of normal human lymphocyte subpopulations.