Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P'1 sites.
Proalbumin is the principal substrate of the in situ hepatic convertase. Here we investigated the specificity of furin using synthetic peptides based on the N-terminal sequence of human proalbumin. The propeptide was rapidly cleaved from the normal ((-6)RGVFRR(-1)DAHKSEAVW(+9)) peptide but as expected, there was no cleavage of the proalbumin Lille analogue with a -2 His (-2H). Surprisingly, the effect of this substitution could not be corrected by introducing a -4 Arg (-4R-2H). In contrast, the peptide -4R-2A was an excellent substrate being cleaved five times faster than normal, indicaabting that His is not allowed as an P2 residue. Replacement of the -4 Val by Glu supported the expected importance of a positive charge at P4 as the cleavage rate dropped to 10% of normal after this substitution. The -6 Arg makes a small contribution to cleavage, its replacement by Ala decreased the cleavage rate to 60% of normal. The Lys-Arg propeptide was almost as good a substrate as the normal Arg-Arg peptide, but the introduction of a Lys at P1 totally abolished processing. The exclusion of P'1 positive charges would be an important requirement for preventing aberrant cleavage in the middle of tetrabasic sequences.
Molecular Pathology Laboratory, Clinical Biochemistry Christchurch Hospital, New Zealand.
S O
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Brennan
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Nakayama
D000595:Amino Acid Sequence; D001779:Blood Coagulation Factors / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism; D045683:Furin; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011228:Prealbumin / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D001779:Blood Coagulation Factors; D001798:Blood Proteins; D010446:Peptide Fragments; D011228:Prealbumin; D011498:Protein Precursors; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D003165:Complement System Proteins; D013381:Subtilisins; D045683:Furin