Application of the multiple antigenic peptides (MAP) strategy to the production of prohormone convertases antibodies: synthesis, characterization and use of 8-branched immunogenic peptides.
Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediately following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84-99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, 1H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventional method of peptide-carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the case of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.
Basak
A
A
Neuropeptides Structure and Metabolism Laboratory, Clinical Research Institute of Montréal, Québec, Canada.
Boudreault
A
A
Chen
A
A
Chrétien
M
M
Seidah
N G
NG
Lazure
C
C
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000917:Antibody Formation; D000941:Antigens / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D002219:Carbamates / Q000737:chemistry / Q000276:immunology; D002234:Carbodiimides; D002851:Chromatography, High Pressure Liquid; D045683:Furin; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011817:Rabbits; D013381:Subtilisins / Q000737:chemistry / Q000276:immunology
D000596:Amino Acids; D000941:Antigens; D002219:Carbamates; D002234:Carbodiimides; D009842:Oligopeptides; D010446:Peptide Fragments; C075359:tetrapeptide carbamate; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases