Membrane peptidases are a multifunctional group of ectoenzymes that have been implicated in the control of growth and differentiation of many cellular systems. Here, using aminopeptidase N/CD13 as an example, Dagmar Riemann and colleagues discuss the role of cell-cell contact in peptidase regulation and the influence of peptidases on cellular functions.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondria1 pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex.2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 -6.1 ascertained by analytical isoelectric focusing.3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 -7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30 -40 0 4 at pH 7.0.5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor.6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.Knowledge of the proteolytic enzymes in the cell is one of the preconditions in studying the molecular mechanism of intracellular protein breakdown. All organelles contain proteolytic activity in various amounts [l]. The lysosomes, in particular, are rich in proteinases. A cell fraction enriched in lysosomes showed at pH 3-4 as well as at pH 6-7 the highest activity hydrolyzing cell-derived proteins in comparison to all other cell organelles [l -61. From this finding it was to be concluded, that the lysosomes certainly play an important role in the overall process of intracellular proteolysis also at physiological pH. A wide variety of lysosomal proteinases has been investigated : cathepsin A (lysosomal carboxypeptidase A) [7-131, cathepsin B1 [7,12-201, cathepsin B2 (lysosomal carboxypeptidase B) [7,12 -14,20-221, cathepsin C (dipeptidyl aminopeptidase I) [7,12,13, Dedicated to Professor Horst Hanson on the occasion of his 65th birthday.Ahbveviation. Leu-CH2C1, 1 -chloro-3-amino-5-methyl-~-hexan-2-one; Tos-Lys-CH2C1, 7-amino-l-chloro-3-tosylamido-~-heptan-2-one ; Tos-Phe-CHzC1, l-chlor0-4-phenyl-3-tosylamido-~-butan-2-one; Bz-L-Arg-NHZ, or-N-benzoyl-L-arginine amide.Enzymes. Cathepsin L (EC 3.4.22.-); cathepsin B1 and B2 (EC 3.4.22.1); cathepsinc (EC 3.4.14.1); cathespin D (EC 3.4.23.5). 18,23,24],cathepsin D [6,7,12,13,23,25-311, cathepsin E [7,13] In studies on the proteolytic activity in a lysosomal extract we succeeded in separating cathepsins BI, C and D by gel filtration on Sephadex G-75. With cytosol proteins as well as with azocasein as substrates at pH 6 and 7, the main part of proteolytic activity was shown to be present in the protein fraction with molecular weights between 20000 and 30000 [2,43]. We could show that in addition to cathepsin B1, the...

Key words: autotaxin; lysophospholipase D; thyroid carcinoma; motilityAutotaxin (ATX) is a 125 kDa glycoprotein that was originally isolated from the conditioned medium of human melanoma A2058 cells. ATX stimulates random and directed motility in a variety of tumor cell lines, including those from prostate and breast carcinomas, melanomas, neuroblastomas and renal-cell carcinoma. [1][2][3][4] Sequence analysis of ATX revealed significant homology to a family of cell surface type I phosphodiesterases, 5 and ATX has recently been newly classified as a member of the ecto-nucleotide pyrophosphate/phosphodiesterase family (E-NPP). Three structurally related mammalian E-NPPs are known, NPP1 (PC-1), NPP2 (ATX, PD-I␣) and NPP3 (gp130 RB 13-6 , PD-I␤, B10). Members of the E-NPP family are capable of hydrolyzing phosphodiester bonds of nucleotides and nucleic acids and pyrophosphate bonds of nucleotides and nucleotide sugars. In addition, they can hydrolyze 3Ј,5Ј-cAMP to AMP, nucleoside 5Ј-triphosphates, such as ATP, to AMP and 2xP i , nucleoside 5Ј-diphosphates, such as ADP, to AMP and P i , or NAD ϩ to AMP and nicotinamide mononucleotide. 6 Recently, ATX (NPP-2) was also shown to hydrolyze lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). 7,8 Active phosphodiesterase, lyso-phospholipase D (lyso-PLD) and migration-stimulating activity of ATX depends on the presence of 4 defined amino acid residues, threonine 210 and histidines 316, 360 and 475, within the ATX molecule. 9 When comparing the substrate specificity of ATX with that of the related family members NPP1 and NPP3, only NPP2 displayed a lyso-PLD activity, whereas NPP1 and NPP3 had a much higher nucleotide pyrophosphatase activity. 10

SUMMARY Interleukin‐17 (IL‐17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL‐17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared to patients with osteoarthritis. In contrast to the restricted expression of IL‐17, the IL‐17 receptor (IL‐17R/CDw217) is expressed ubiquitously. Using a real‐time RT‐PCR assay, we detected similar absolute levels of IL‐17R mRNA expression in fibroblast‐like synoviocytes (SFC) from patients with RA (mean 9 pg/μg total RNA; ranged from 0·1 pg to 96 pg IL‐17R mRNA/μg total RNA) compared to synoviocytes of non‐RA patients. Analysis of the IL‐17R surface expression confirmed the results obtained for IL‐17R mRNA expression. Exposure of SFC to IL‐17 led to a mRNA induction of CXC chemokines IL‐8, GRO‐α and GRO‐β. An anti‐IL‐17 antibody blocked these effects of IL‐17. The MAPK p38 appears necessary for the regulation of IL‐8, GRO‐α and GRO‐β expression as shown by inhibition with SB203580. The inhibitors genistein (tyrosine kinase inhibitor) and calphostin C (inhibitor of protein kinase C) reduced significantly the IL‐17‐stimulated mRNA expression of IL‐8, GRO‐α and GRO‐β in SFC, whereas PD98059 (inhibitor of MEK‐1/2) was without effect. Pharmacological drugs used in therapy of RA, such as cyclosporin and methotrexate, induced a fourfold increase of IL‐17R mRNA expression and augmented the IL‐17‐stimulated IL‐8 expression. Our results support the hypothesis that IL‐17/IL‐17R may play a significant role in the pathogenesis of RA contributing to an unbalanced production of cytokines as well as participating in connective tissue remodelling.

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