Furin, PC1/3, and/or PC6A Process Rabbit, but Not Human, Pro-lactase-phlorizin Hydrolase to the 180-kDa Intermediate

Patrick Keller,Laura Zecca,Rita Boukamel,Elmar Zwicker,Sergio Gloor,Giorgio Semenza

doi:10.1074/jbc.270.43.25722

Patrick Keller, Laura Zecca + Show 4 more

Open Access

https://doi.org/10.1074/jbc.270.43.25722

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Abstract

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.

Highlights

Hydrolase) [3], the membrane-spanning segment, and the cytosolic C terminus make the “mature” lactase-phlorizin hydrolase (LPH) which can be isolated from the brush-border membrane
Antibodies against a peptide corresponding to the 59 N-terminal amino acid residues of pro-LPH, which did precipitate pro-LPH, failed to precipitate the 180-kDa intermediate form and mature LPH (Fig. 1B, lane Ia); the intermediate form was instead precipitated by antibodies raised against a later segment of region I and a segment of region II
Our conclusions are that rabbit enterocytes process pro-LPH to the 180-kDa intermediate by way of furin and possibly PC1/3 and/or PC6A for the following reasons

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals were of the highest possible purity and were purchased from Fluka (Buchs, Switzerland) unless otherwise indicated. 72 h after transfection, the cells were washed with MEM, and incubated in 2 ml of labeling medium (MEM supplemented with 10% dialyzed FCS) for 60 min at 37 °C and 5% CO2. In the first reaction (67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 6.7 mM MgCl2, 10 mM 2-mercaptoethanol, 1 mM dNTP, 10% dimethyl sulfoxide, 0.72% bovine serum albumin, and 1.2 ␮M concentration of each primer), primers SGrlac3ϩ and SGrlac4Ϫ were used and samples were taken through 35 cycles of 1 min at 94 °C, 90 s at 40 °C, and 90 s at 65 °C This reaction produced a 600-base pair DNA fragment. RNA probes were synthesized in the presence of biotin-11-UTP (Boehringer) from the rabbit cDNA clones described above The sizes of these cRNAs were approximately 600, 400, 600, and 600 nucleotides for furin, PC1/3, PC2, and PC6A/B, respectively. The slides were removed from the machine and slightly counterstained with Mayer’s hemalum solution (Merck) prior to mounting with aquamount solution (BDH)

RESULTS

DISCUSSION

Enterocytesa Enterocytes