Furin, PC1/3, and/or PC6A process rabbit, but not human, pro-lactase-phlorizin hydrolase to the 180-kDa intermediate.
Small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a large precursor (prepro-LPH) of 1926 amino acids. In the endoplasmic reticulum, prepro-LPH is split by signal protease. The resulting pro-LPH is cut to mature LPH directly (human) or via a 180-kDa intermediate (rabbit), most likely in the trans-Golgi network or in a later compartment. Antibodies directed against different regions of rabbit pro-LPH locate the cleavage site resulting in the 180-kDa intermediate between amino acid residues 79 and 286. This stretch contains the two sequences -Arg-Cys-Tyr-Arg114 approximately -Arg-Ala-Ser-Arg191 approximately, which are potential cleavage sites for subtilisin-like proprotein convertases. These sites are not conserved in human pro-LPH. By coexpression in COS 7 cells of rabbit prepro-LPH and proprotein convertases (PC 1/3, PC2, PC6A, PC6B, furin), we show that furin, PC 1/3, and PC6A generate a processing intermediate that is immunologically indistinguishable from the one observed in vivo. Furin, PC 1/3, and PC6A are all expressed in the small intestine as shown by a polymerase chain reaction-based approach and, more specifically, in enterocytes, as shown by in situ hybridization. These results suggest that furin, PC 1/3, and/or PC6A are responsible for the in vivo processing of rabbit pro-LPH to the 180-kDa intermediate.
Keller
P
P
Department of Biochemistry, Swiss Federal Institute of Technology, ETH Zentrum, Zurich, Switzerland.
Zecca
L
L
Boukamel
R
R
Zwicker
E
E
Gloor
S
S
Semenza
G
G
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D020126:Brefeldin A; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D045683:Furin; D006801:Humans; D017403:In Situ Hybridization; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010694:Lactase-Phlorizin Hydrolase / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D016133:Polymerase Chain Reaction; D045664:Proprotein Convertase 1; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D014018:Tissue Distribution; D014162:Transfection
D003517:Cyclopentanes; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C096385:pro-lactase-phlorizin hydrolase; D020126:Brefeldin A; D010694:Lactase-Phlorizin Hydrolase; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; D045683:Furin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases