Related Experiment Videos
Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage
Eric Bergeron1, Ajoy Basak, Etienne Decroly
1Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC, Canada, H2W 1R7.
Insights
Proprotein convertases (PCs), including furin and PC5A, cleave integrin alpha4. This cleavage, crucial for alpha4 function, occurs in acidic cellular compartments and is influenced by a P6 histidine residue.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Proprotein convertases (PCs) are essential enzymes involved in the post-translational modification of numerous proteins.
- Integrin alpha4 plays a critical role in cell adhesion and migration, and its proper processing is vital for its function.
Purpose of the Study:
- To identify the specific proprotein convertases responsible for cleaving the integrin alpha4 subunit.
- To elucidate the molecular determinants and cellular conditions governing integrin alpha4 processing.
Main Methods:
- Co-expression of alpha4 with various PCs in furin-deficient LoVo cells.
- Analysis of endogenous pro-alpha4 processing in CEM-T4 cells overexpressing PCs.
- Site-directed mutagenesis of the alpha4 cleavage site.
- In vitro kinetic studies with synthetic peptides.
- Biosynthetic analysis using NH(4)Cl treatment.
Main Results:
- Furin and proprotein convertase 5A (PC5A) were identified as the primary convertases for pro-alpha4.
- Cleavage is dependent on a positively charged residue at P1 (Arg597) and influenced by a histidine at P6 (His592).
- Pro-alpha4 cleavage by furin is pH-sensitive, suggesting it occurs in acidic cellular compartments.
Conclusions:
- The study identifies furin and PC5A as key enzymes for integrin alpha4 processing.
- The findings extend the known furin processing motif to include a regulatory P6 histidine, crucial for pH-dependent cleavage.
- This suggests that integrin alpha4 processing occurs preferentially in acidic endosomal or lysosomal compartments.
Abstract:
The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 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-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) 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 alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.