Molecular cloning and expression of cDNA for human antileukoprotease from cervix uterus

Abstract

We have isolated cDNA clones for the human antileukoprotease HUSI-I, an elastase inhibitor, from a library, containing cDNA inserts made from human cervix uterus. A library of 10000 recombinants was screened using a mixture of 16 different oligodeoxyribonucleotides which correspond to amino acids 79 -84 and one 20mer oligodeoxyribonucleotide corresponding to amino acids 19 -26. Two overlapping cDNA clones, containing the entire coding sequence and part of the 5'-and 3'-untranslated region, were isolated. DNA sequence data showed that our clone corresponds with the available protein sequence data. For expression, the cDNA fragment was inserted in a derivative of plasmid pPLc236 and expressed under the control of APL promoter. Expression of antileukoprotease was proven by Western blot analysis and inhibition of chymotrypsin.Neutral granulocytic proteinases as well as kallikreins are known to be potent mediators of the inflammatory response following polytraumatic injuries [I, 21. In vivo inhibition of these proteinases by low-molecular-mass inhibitors could serve as a valuable tool to decrease the inflammatory reaction [l, 3,4]. Human mucous fluids such as seminal plasma, cervical mucus, tears, bronchial and nasal secretions contain acidstable proteinase inhibitors with strong affinity for trypsin and chymotrypsin as well as for neutrophil lysosomal elastase and cathepsin G [5, 61. These inhibitors are characterized by their acid stability and low molecular mass. The antileukoprotease from human seminal plasma HUSI-I (human seminal plasma inhibitor I) and the inhibitor found in mucous secretions, from the cervix uteri, although isolated from different tissues seem to be identical proteins based on shared characteristics of acid stability and low molecular mass [I], partial amino acid sequence homology [7], inhibition spectrum and immuno-cross-reactivity [S]. We shall therefore refer to them jointly as ALP (antileukoprotease). The isolation of the inhibitor from human seminal plasma is complicated

Inhibitory Function of Whey Acidic Protein in the Cell-Cycle Progression of Mouse Mammary Epithelial Cells (EpH4/K6 Cells)

“…WAP proteins have a signal peptide [11] and two domain structures that are identified at the fourdisulfide core (4-DSC) domain, which is composed o f ei g h t c y s t ei n e r es id ue s i n a c o ns er v ed arrangement [12]. The 4-DSC domain arrangement is not exclusive to the WAP family, and a number of other proteins containing 4-DSC domains have been identified as protease inhibitors [11,[13][14][15][16], comprising either single or multiple copies of the 4-DSC domain. Generally, these proteins are secreted and may function as protease inhibitors [17,18].…”

Secretory Leukocyte Protease Inhibitor Mediates Proliferation of Human Endometrial Epithelial Cells by Positive and Negative Regulation of Growth-associated Genes

“…The carboxyl-terminal domain manifests inhibitory activities against chymotrypsin, trypsin, granulocyte and pancreatic elastases, cathepsin G, and mast cell chymase (3,4). SLPI is expressed primarily in secretory/glandular epithelial cells of a variety of human tissues including the bronchi, parotid glands, small and large intestines, skin, breast, pancreas, male and female genital tracts, and kidney (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Its relative absence in serum, except in certain pathological conditions (15,16), and its localization to extracellular matrix and subcellular sites not readily accessible to larger molecular weight protease inhibitors such as ␣ 1 -antitrypsin (17), suggests a primarily autocrine/paracrine mode of action.…”

Whey Acidic Protein (WAP) Depresses the Proliferation of Mouse (MMT) and Human (MCF-7) Mammary Tumor Cells

“…WAPs have a signal peptide [14] and two domain structures with a four-disulfide core (4-DSC) in a conserved arrangement [15]. The 4-DSC domain arrangement is common in the WAP family, and many other proteins containing 4-DSC domains have been identified as protease inhibitors [14,[16][17][18][19], comprising either single or multiple copies of the 4-DSC domain.…”