The proprotein convertases (PCs) participate in the limited proteolysis of integrin α4 subunit at the H592VISKR597 ↓ ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and ↓ indicates the cleavage site), since this cleavage is inhibited by the serpin α1-PDX (α1-antitrypsin Portland). Co-expression of α4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-α4 convertases. In agreement, processing of endogenous pro-α4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-α4 convertase. Mutational analysis showed that replacement of P1 Arg597 with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type α4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-α4 and its H592R and H592K mutants in the presence or absence of the weak base, NH4Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-α4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg↓, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.
Abbreviations used: Abz, 2-aminobenzoic acid; AMC, 7-amino-4-methylcoumarin; BTMD, before transmembrane domain; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; ER, endoplasmic reticulum; EGFP, enhanced green fluorescence protein; endoH, endoglycosidase-H; FBS, foetal bovine serum; HRP, horseradish peroxidase; mAb, monoclonal antibody; MCA, 4-methylcoumarin-7-amide; MALDI–TOF-MS: matrix-assisted laser-desorption ionization–time-of-flight MS; PACE, paired basic amino acid cleaving enzyme; PC, proprotein convertase; α1-PDX, α1-antitrypsin Portland; QFP, quenched fluorogenic peptide; RP-HPLC, reverse phase HPLC; RT, reverse transcriptase; TGN, trans-Golgi network; Tyx-A, 3-nitro-Tyr-Ala; VCAM-1, vascular cell adhesion molecule-1; VV, vaccinia virus; WT, wild-type.
Present address: INSERM U372, Pathogénie des Infections à Lentivirus, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13276 Marseille, Cedex 9, France.