Literature summary for 3.4.21.75 extracted from

Mamedov, T.; Musayeva, I.; Acsora, R.; Gun, N.; Gulec, B.; Mammadova, G.; Cicek, K.; Hasanova, G.
Engineering, and production of functionally active human furin in N. benthamiana plant in vivo post-translational processing of target proteins by furin in plants (2019), PLoS ONE, 14, e0213438 .

Search for more literature for 3.4.21.75

Cloned(Commentary)

Cloned (Comment)	Organism
functional recombinant expression of His6-tagged human furin lacking its signal peptide in Nicotiana benthamiana plants. Plant-produced human furin is highly active both in vivo and in vitro and specifically cleaves the tested target proteins, factor IX (FIX) and protective antigen (PA83). Plants lack some of the important posttranslational modifications, including furin processing, but both, enzymatic deglycosylation and proteolytic processing of target proteins, can be achieved in vivo by coexpression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. Recombinant coexpression of engineered enzyme and substrate using the Agrobacterium tumefaciens strain AGL1 transfection method, expression in leaves. Coexpression of human furin with PA83 of Bacillus anthracis and deglycosylation enzymes Endo H or PNGase F in Nicotiana benthamiana. Deglycosylation efficiency of plant produced Endo H is greater than that of plant produced PNGase F. When full length furin is coexpressed with the PA83 protein in Nicotiana benthamina plants, there is little or no cleavage of PA83. Plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity	Homo sapiens

Cloned (Comment)

Organism

functional recombinant expression of His6-tagged human furin lacking its signal peptide in Nicotiana benthamiana plants. Plant-produced human furin is highly active both in vivo and in vitro and specifically cleaves the tested target proteins, factor IX (FIX) and protective antigen (PA83). Plants lack some of the important posttranslational modifications, including furin processing, but both, enzymatic deglycosylation and proteolytic processing of target proteins, can be achieved in vivo by coexpression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. Recombinant coexpression of engineered enzyme and substrate using the Agrobacterium tumefaciens strain AGL1 transfection method, expression in leaves. Coexpression of human furin with PA83 of Bacillus anthracis and deglycosylation enzymes Endo H or PNGase F in Nicotiana benthamiana. Deglycosylation efficiency of plant produced Endo H is greater than that of plant produced PNGase F. When full length furin is coexpressed with the PA83 protein in Nicotiana benthamina plants, there is little or no cleavage of PA83. Plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
additional information	engineering of the human furin gene for expression in plants, the production of a functional active recombinant truncated human furin in Nicotiana benthamiana plants. The signal peptide (amino acids 1-25) is removed from the furin sequence, and Nicotiana tabacum PR-1a signal peptide (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) is added to the N-terminus. The KDEL sequence (the endoplasmic reticulum retention signal) and the His6 tag (the affinity purification tag) are added to the C-terminus. A truncated form of furin (amino acids 26-595) is expressed with a PR-1a signal peptide at N-terminus and His6-KDEL at C-terminus	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	the enzyme contains a signal peptide (amino acids 1-25) and is secreted	Homo sapiens	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	required	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
factor IX + H2O	Homo sapiens	-	?	-	?
PA83 + H2O	Homo sapiens	a protective antigen	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P09958	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	N-glycosylation is necessary for the proper folding and catalytic activity of plant produced or commercial human furin (NEB). Plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity. Similarly, when commercial human furin (NEB) is treated with commercial Endo H or PNGase F, no cleavage is observed for the substrate PA83	Homo sapiens

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Nicotiana benthamiana leaves by nickel affinity chromatography, co-purification of the coexpressed proteins	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
factor IX + H2O	-	Homo sapiens	?	-	?
additional information	to transiently express the substrate factor IX (FIX, amino acids 29-461) in Nicotiana benthamiana plants, the signal peptide (amino acids 1-28) is removed from the FIX sequence (UniProt ID P00740) and replaced with a Nicotiana tabacum PR-1a signal peptide. a KDEL sequence and the His6 tag are added to the C-terminus. Enzyme substrate precursor polypeptide of factor IX (FIX) undergoes several post translational modifications (PTMs), including the removal of the signal peptide (aa 1-28), carboxylation of the first 12 glutamic acid residues downstream from the 18-amino acid propeptide sequence (aa 29-46) in the region rich in glutamic acid (aa 47-92, called the gamma-carboxyglutamic acid or Gla domain) at the N-terminus. Proper gamma-carboxylation of the Gla domain is required for binding to calcium and phospholipids that is critical for proper protease activity during coagulation. In vivo, vitamin K-dependent gamma-carboxylase binds to the 18-amino acid propeptide of FIX, which is then cleaved and is required for optimal binding of the Gla domain to Ca2+ and phospholipids	Homo sapiens	?	-	?
PA83 + H2O	the protective antigen substrate is from Bacillus anthracis, commercial plant-produced deglycosylated PA83 (dPA83). Cleavage of the substrate generates two protein fragments with distinctly different molecular masses of 63 kDa (PA63) and 20 kDa (PA20). Plant-produced PA83 protein is almost fully cleaved by commercial furin, while plant-produced truncated enzymatically active furin displays about 75% relative activity compared to commercial human furin in vitro	Homo sapiens	PA63 + PA20	-	?
PA83 + H2O	a protective antigen	Homo sapiens	?	-	?
protein APRIL + H2O	commercial substrate preparation	Homo sapiens	?	-	?

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Homo sapiens

General Information

General Information	Comment	Organism
malfunction	plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity. Similarly, when commercial human furin (NEB) is treated with commercial Endo H or PNGase F, no cleavage is observed for the substrate PA83	Homo sapiens
physiological function	furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events	Homo sapiens