α 1 -Antitrypsin: The Presence of Excess Mannose in the Z Variant Isolated from Liver

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The cellular defect in alpha 1-proteinase inhibitor (alpha 1-PI) deficiency is expressed in human monocytes and in Xenopus oocytes injected with human liver mRNA.

Perlmutter¹,

Kay²,

Cole³

et al. 1985

“…This is the case even in the presence of NH4C1, which enhances the secretion of most newly formed lysosomal enzymes (2,23 (27). In one type of a1-antitrypsin deficiency, an insoluble variant form of antitrypsin aggregates and accumulates in the endoplasmic reticulum (28,29). Some virus mutants blocked in G-protein maturation (30) and an altered HLA-A2 antigen of mutant lymphoblastoid cells (31) fail to reach the cell surface.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of beta-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor alpha chain in a rare form of Tay-Sachs disease.

Proia¹,

Neufeld²

1982

104

RNA was isolated from human term placenta or cultured fibroblasts and translated in a rabbit reticulocyte system in the presence of [3S]methionine; the translation products were immunoprecipitated with antisera made against 3-hexosaminidase or its isolated a and ( chains and analyzed by polyacrylamide gel electrophoresis. The largest translated a and (3chain polypeptides had Mrs of 65,000 and 59,000, respectively. These are a2,ooo greater than the Mrs of precursor chains synthesized by intact fibroblasts and deglycosylated with endo-,3-N-acetylglucos- , ( Hexosaminidase is a lysosomal enzyme relevant to several heritable diseases. The A isozyme of f3-hexosaminidase is composed of a and , subunits, which are structurally dissimilar and encoded on different chromosomes; the B isozyme is composed of (3 subunits only. Tay-Sachs disease affects the a subunit and, therefore, the A isozyme; Sandhoffdisease affects the (3(or common) subunit and, therefore, both isozymes A and B. A third disease entity (AB variant) allows the production of normal (3-hexosaminidase but not ofits activator. These diseases are characterized by marked accumulation of GM2 ganglioside, the major natural substrate of ,B-hexosaminidase A, resulting in progressive loss of nervous system function; they are usually fatal in early childhood. Variant forms of later onset and reduced severity are also known. The clinical, genetic, and biochemical aspects ofthesedisordershavebeen reviewed(1).We previously have studied the synthesis and processing of the two chains of (3-hexosaminidase in cultured human skin fibroblasts (2-4). 6360The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…Inherited a1-antitrypsin deficiency occurs at a high frequency in European populations (1 in 750 for the two principal variants Z and S) (4). The most common clinically significant variant (type Z) has a single amino acid substitution that is associated with reduced glycosylation of the a1-antitrypsin molecule (5)(6). This results in its accumulation in hepatocytes and a reduction in serum concentration to 10-15% of normal (7).…”

Section: Introductionmentioning

confidence: 99%

High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

Courtney¹,

Buchwalder²,

Tessier³

et al. 1984

124

A cDNA clone containing the complete human a1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the a1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage A (PL) and initiation of translation at the A cli gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human a1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.a1-Antitrypsin is a serum antiprotease of hepatic origin whose most important physiological role is to restrict neutrophil elastase activity in the lung (1-2). A deficiency of a1-antitrypsin upsets the alveolar protease-antiprotease balance, leading to elastase-mediated tissue destruction and chronic pulmonary emphysema (3). Inherited a1-antitrypsin deficiency occurs at a high frequency in European populations (1 in 750 for the two principal variants Z and S) (4). The most common clinically significant variant (type Z) has a single amino acid substitution that is associated with reduced glycosylation of the a1-antitrypsin molecule (5-6). This results in its accumulation in hepatocytes and a reduction in serum concentration to 10-15% of normal (7). Cigarette smoking, a major factor in nonhereditary emphysema, also causes a protease-antiprotease imbalance in the lower respiratory tract (8) as a consequence of both increased elastase levels and a 50% reduction in active alveolar a1-antitrypsin (9)(10). Clinical trials have shown that a1-antitrypsin deficiency can be treated by replacement therapy (11), but the problems of possible viral contamination associated with the use of human blood products deter extensive clinical use of a1-antitrypsin purified from serum. To circumvent this problem we have used the techniques of genetic engineering to produce human a1-antitrypsin in a microorganism. The availability of information on the sequences of baboon and human a1-antitrypsin cDNA clones (12, 13) and the structures of normal and variant genes (14, 15) enabled us to isolate a full-length human a1-antitrypsin cDNA clone. Transfer of this sequence into a high-level expression system resulted in a recombinant E. coli strain capable of synthesizing a1-antitrypsin at levels of up to 15% of total cell protein. MATERIALS AND METHODSBacterial Strains and Plasmids. cDNA banks were prepared using E. coli strain 1106 (supE hsdS met supF). Strain TGE900 [F-su-ilv-bio (XcI857ABamAHI)], which produces the temperature-sensitive XcI857 repressor, was used as host for the PL-containing plasmids.pTG603 is a pBR322 derivative containing a human a1-antitrypsin cDNA insert. pTG920 is the PL-containing expression vector and pTG922 is a derivative that expresses human a1-antitrypsin.Isolation of a1-...