Partial purification and some properties of a new papain inhibitor from psoriatic scales.

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum.

Antisense RNA inhibition of cathepsin L expression reduces tumorigenicity of malignant cells.

Several tumour-forming cell lines are known to overproduce the lysosomal cysteine peptidase cathepsin L. We have used an antisense approach to investigate whether inhibition of cathepsin L overexpression in two malignant cell lines (myeloma SP cells and L cells) reduces their tumorigenic potential. Two different cDNA fragments of murine cathepsin L were inserted in the antisense direction into the pcDNA3 vector, and SP and L cells were stably transfected with these plasmid constructs. Several of the selected clones expressing the antisense transcript showed specific reduction of the mRNA level and the intracellular activity of cathepsin L, and a greatly diminished amount of secreted procathepsin L. When tested in Balb/c nu/nu mice, the cell lines with low cathepsin L activity exhibited a significantly decreased potential for tumour growth when compared with control cells expressing wild-type levels of cathepsin L activity. This observation suggests that cathepsin L is a critical factor in tumour growth.

Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay.

A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum.

Expression of the 43 kDa papain inhibitor during human fetal skin development.

The presence of the 43 kDa papain inhibitor protein in the skin of 40 fetuses with the gestational age varying from 9 to 40 weeks was studied. Immunohistochemistry showed the first evidence of the 43 kDa papain inhibitor in the acral parts, mostly in the nail bed, at the 14th week when the epidermis in all other sites was not stained. Staining of the follicular infundibulum as well as some parts of the uppermost epidermis and keratin layer was observed from 17 to 40 weeks. The staining was most intensive at 20-30 weeks gestational age, and later the intensity gradually decreased so that in the full-term newborn the 43 kDa papain inhibitor was hardly detectable or absent. It was concluded that the 43 kDa papain inhibitor is present in fetal skin at the stage of stratification (9-14 weeks) only in the nail region and its volar surroundings. It appears at the stage of interfollicular keratinization (14-24 weeks) in the uppermost epidermis, especially in the appendical openings and its amount seems the decrease during the stage of interfollicular keratinization (24 weeks--full-term). The expression of 43 kDa inhibitor protein is temporally related to that of filaggrin.

Inactive cathepsin B-like enzyme in human melanoma culture medium.

An inactive cathepsin B-like enzyme with a molecular mass of 40 kD was found in a human melanoma culture medium. The inactive form of this enzyme was converted into an active form with a molecular mass of 28 kD by pepsin treatment. This activated cathepsin B-like enzyme had almost the same characteristics regarding molecular size, substrate specificity, dependence on chemical reagents, and Km values as intracellular cathepsin B. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by electroblotting with an antiserum against cathepsin B yielded inactive cathepsin B-like enzyme fractions which showed two immunoreactive bands with molecular masses of 40 and 28 kD, respectively. On the other hand, alkali treatment of the inactive cathepsin B-like enzyme fractions released a cysteine proteinase inhibitor with a molecular mass of 12 kD. These data suggest that these inactive cathepsin B-like enzymes in melanoma culture medium are present not only in the precursor form, but that they are also present as enzyme-inhibitor complexes, both of which can be activated enzymatically in vitro.

Separation and partial characterization of four cysteine proteinases from a human epidermal cell line.

Four different cysteine proteinases from a cultured human epidermal cell line (NCTC 2544) were partially purified and characterized. The biggest hydrolase was an endoaminopeptidase with the molecular weight of several hundred kilodaltons. It was a glycoprotein and had an almost neutral pH optimum. The three other hydrolases resembled lysosomal cathepsins B, H, and L in various respects except for somewhat higher molecular weight for cathepsin B (29 kDa) and the cathepsin H-like (70 kDa) hydrolase than those reported from most other tissues. Low molecular weight cysteine proteinase inhibitors ACPI (cystatin A) and NCPI (cystatin B) inhibited the cathepsins, but not the high molecular weight proteinase.

Plasminogen activators of psoriatic scale extracts. Separation of two plasminogen activators by isoelectric focusing.

Psoriatic scale extracts were fractioned by using polyacrylamide gel isoelectric focusing (PAGIF) and preparative electrofocusing in granulated gel (PEGG). The largest protein fraction was found with Ip at pH 4.8--5.0, and the main protein bands within pH values 4.0--7.5. PEGG separated three main fractions with plasminogen activator or trypsin-like esterase activity with isoelectric points at pH 6.5--6.6, 5.4--6.2 and 4.9. The enzyme with Ip at pH 6.5--6.6 hydrolyzed trypsin substrates but lacked plasminogen activator capacity. The enzyme with Ip at pH 5.4--6.2 showed both activities but the third enzyme with plasminogen activator capacity with Ip at pH 4.9 was without detectable esterolytic activity towards substituted basic amino acid esters. The third enzyme was prominent in KCl-extract and the second in KSCN-extract. The first was equal in both extracts. The enzyme with Ip at pH 4.9 is possibly of bacterial origin while the plasminogen activator with Ip at pH 5.4--6.2 extracted in KSCN probably represents tissue activator of psoriatic scales.

Proteinase binding and enhancing factor from human skin.

Fresh human skin extract made in salt solution after a prior buffer extraction was shown to enhance the hydrolysis of N-alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) by trypsin. This trypsin enhancing effect was further shown to be both stabilizing and activating. After chromatography on Sephadex G-100, the trypsin binding factor was found in fractions of void volume. Protease binding took place in physiological and hypotonic but not in hypertonic NaCl-solutions (0.5 mol/l). The proteinase binding factor was further purified by trypsin-Sepharose 4 B affinity chromatography. It was found to bind also chymotrypsin and elastase and to be thermostable (100 degrees C for 20 min), precipitable at acidic pH (3.5), and by acetone and ammonium sulphate (60% saturation). The bound proteinases were found to preserve their hydrolytic activity towards protein substrates. Bound trypsin and chymotrypsin could completely be inhibited by soybean trypsin inhibitor. The binding factor did not react with anti-human-alfa2-macroglobulin antiserum from rabbit.

A 43-kDa papain inhibitor produced by a cultured human epidermal cell line.

Epidermis-derived cells (NCTC 2544) were cultured and the proteins of the culture medium, as well as of the cells, were fractionated by gel-chromatography. The fractions were analyzed for their papain-inhibitory capacity and for the presence of so-called 43-kDa papain inhibitor. A papain inhibitor was identified with molecular weight and immunological characteristics similar to the original 43-kDa inhibitor that was isolated from psoriatic scales. The result proves that NCTC-2544 cells can produce the so-called psoriasis inhibitor under culture conditions.

Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line.

Human skin epithelial-like cells (NCTC-strain 2544) were grown in RPMI-1640 medium supplemented with foetal calf serum for up to 2 weeks. The culture medium and extracts made from the cells were subjected to gel-filtration chromatography in a Sephacryl S-200 column for fractionation of the proteins. The fractions were assayed for acid and neutral cysteine-proteinase inhibitor (ACPI, NCPI) using time-resolved fluoroimmunoassay and radioimmunoassay, and the cysteine-proteinase-inhibiting activities were assayed using papain. Free NCPI, i.e. a molecule with isoelectric variants at pHs 6.0 and 6.5, which has an Mr of around 12,000 and is capable of inhibiting papain, was detected both in the culture medium and in the cells. Immunodiffusion studies revealed its immunological identity with human spleen-derived NCPI. The amount of NCPI increased during the incubation period. ACPI--characterized as a molecule having an isoelectric point of 4.9, an Mr of about 12,000, papain-inhibiting capacity and antigenic reactivity with spleen-derived ACPI--was not detected in the culture medium. It was, however, detected in the cells after 2 weeks in culture. These data prove that ACPI and NCPI are synthesized by the NCTC-2544 cells under the present culture conditions.

Cysteine proteinase inhibitors in psoriatic epidermis.

Human psoriatic epidermis and scales were demonstrated to contain two antigenically separate cysteine proteinase inhibitors, one acidic with an isoelectric point of 4.7-5.0 (ACPI) and one neutral with an isoelectric point of 6.0-6.5 (NCPI), while normal epidermis contains only ACPI. The total papain (cysteine proteinase) inhibiting activity of the psoriatic epidermis as calculated per mg protein was higher than that in normal epidermis. Both ACPI and NCPI were localized immunocytochemically, mainly in the highest spinous cell layers with less activity in the parakeratotic cells and lower layers of spinous cells. Basal cells were essentially negative.

Purification and characterization of a cystatin-type cysteine proteinase inhibitor in the human hair shaft.

We found a cysteine proteinase inhibitor in human hair shaft extract treated with 0.01 M Tris HCl buffer, pH 8.0. A yield of 0.2 mg of purified cysteine proteinase inhibitor was obtained from 86 g of hair shaft. The cysteine proteinase inhibitor had a molecular mass of 13 kDa as determined by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was more stable to heat and pH than most proteins and had a pI of 4.7. Immunologically, its antigenicity was the same as that of cystatin A, but differed from that of cystatin B and C, and kininogen. The amino-acid sequence of the first 30 residues from the NH terminus of the inhibitor was identical to that of cystatin A from human epidermis. Hair shaft cysteine proteinase inhibitor is thus considered to be identical to epidermal cystatin A.

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia.

The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

A method for the hyaluronic acid synthetase assay in human skin.

A method for the determination of hyaluronic acid synthetase activity of human skin is described. Skin samples were crush-homogenized, and incubated with UDP-14C-glucuronic acid and UDP-N-acetylglucosamine in Tris-HCl-buffer containing MgCl2. After papain digestion of the samples, the 14C-labeled hyaluronic acid was separated in Sephadex G 50 chromatography. The radioactivity incorporated into hyaluronic acid was an expression of the activity of hyaluronic acid synthetase. This activity was found to be increased in skin biopsies obtained from psoriatic lesions and decreased after local treatment with potent corticoids.

A chloride activated alanine aminopeptidase from a melanoma cell line.

Alanine aminopeptidase was partially purified from cultured human melanoma cells (Bowes) by gel filtration on Sephadex G-200 and DEAE Sepharose column chromatography. The molecular weight of the enzyme was about 52,000 as determined by gel filtration on Sephadex G-100. The enzyme hydrolyzed L-alanine beta-naphthylamide (NA), but not or slightly L-methionine-NA, L-leucine-NA, and L-arginine-NA. The Km value for L-alanine-NA was 0.17 mmol/l, pH optimum was 7.4. The enzyme was stable at 50 degrees C for 20 min, but lost about 50% of its activity at 60 degrees C within 20 min. It was markedly stimulated by chloride ions, and was inhibited by sulfhydryl blocking agents and EDTA. The activity was restored by the addition of Co2+ or Zn2+ after EDTA treatment. The enzyme is a metallo- and thiol-dependent and chloride-activated, low-molecular weight aminopeptidase.

An inactive cathepsin B-like enzyme and cysteine proteinase inhibitors in colon cancer ascites.

The ascitic fluid of a patient with colon cancer was found to contain an inactive cathepsin B-like enzyme. The inactive enzyme with a molecular weight of 40 kDa was converted by pepsin treatment into an active form with a molecular weight of 28 kDa as revealed by Sephadex G-75 gel chromatography. The inactive cathepsin B-like enzyme was considered to represent a precursor form and not an enzyme-inhibitor complex. The activated cathepsin B-like enzyme resembled human liver cathepsin B in its enzymatic characteristics. Cysteine proteinase inhibitor activity was also detected in the same ascitic fluid, and it was separated into two main forms by Sephadex G-75 gel chromatography. The high molecular weight inhibitor fractions reacted with antiserum against alpha-CPI and the low molecular weight fractions reacted with antiserum against cystatin B.

Cysteine proteinase inhibitors in human squamous cell carcinoma.

Cysteine proteinase inhibitors in Tris extract of human skin squamous cell carcinoma tissue were studied. From 17.2g of the tissue, approximately equal to 113.5 U inhibitor was obtained. With Sephadex G-75 gel chromatography, 3 papain inhibitor peaks of Mr approximately equal to 100,000, 38,000, and 12,000 were separated. In the immunodiffusion studies, Mr approximately equal to 100,000 fractions reacted with antisera made against human kininogen (alpha-cysteine proteinase inhibitor) and cystatin B. By using alkali treatment, free cystatin B was dissociated from the Mr approximately equal to 100,000 fractions. It is suggested that Mr approximately equal to 100,000 fractions contain cystatin B and alpha-cysteine proteinase inhibitor complexed with tissue proteinases. The Mr approximately equal to 38,000 fractions reacted with antiserum made against the papain inhibitor of Mr approximately equal to 43,000. The inhibitors in the Mr approximately equal to 12,000 fractions were identified as cystatin A and B on the basis of their reactions with antisera made against these inhibitors purified from human tissues.

Human cystatins in normal and diseased tissues--a review.

Immunohistochemica and quantitative immunochemical methods were used to demonstrate the presence of two cysteine proteinase inhibitors, cystatins A and B, in normal and diseased tissues. Cystatin A is expressed in squamous epithelia, neutrophil granulocytes, and dendritic reticulum cells of the lymphatic tissues. Its concentration is increased in inflammatory skin diseases and decreases after the malignization of squamous epithelia. Cystatin B is seen in wet squamous epithelia, and in the cells of monocyte-macrophage series, where its concentration varies depending on the activation state of the cells. In the malignant keratinocytes cystatin B follows the behaviour of cystatin A.

Natural cysteine proteinase inhibitors reduce lectin induced lymphocyte stimulation.

Lymphocyte stimulation by lectins can be inhibited by several synthetic inhibitors of proteolytic enzymes, notably those of cysteine proteinases. The effects of naturally occurring enzyme inhibitors are less well known. The effect of the neutral low-molecular weight cysteine proteinase inhibitor (NCPI) recently purified from lymph nodes and spleen was therefore investigated. Cultured peripheral blood lymphocytes were stimulated by PHA or ConA in the presence or absence of NCPI and the incorporation of 3H-thymidine was measured. NCPI was found to inhibit these lymphocyte responses in these circumstances.

The 43 kDa inhibitor of human skin and other epithelia.

We have used immunohistochemical and quantitative immunochemical techniques to study the tissue distribution of the 43 kDa papain inhibiting protein, originally isolated from psoriatic scale. High amounts of the inhibitor were found in the epidermis only in several skin diseases where the epidermal cell proliferation was increased (psoriasis, various eczemas). The inhibitor was also found in the basal cells of the bronchial epithelium and in the squamous epithelia of mouth and oesophagus, and the Hassal's corpuscles of the thymus. Sera of patients suffering from several skin diseases did not contain the inhibitor, but antibodies against it were found in the sera of two patients suffering from leg ulceration. The inhibitor was immunologically unrelated to filaggrin, involucrin, or keratins.