Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.
 ...
PMID:Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases. 866 47

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.
 ...
PMID:Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures. 882 70

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.
 ...
PMID:Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line. 1279 Aug 3