Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family.
The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.
Wuebbens
M W
MW
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Roush
E D
ED
Decastro
C M
CM
Fierke
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology; D015153:Blotting, Western; D002257:Carbonic Anhydrase Inhibitors / Q000097:blood / Q000737:chemistry* / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015151:Immunoblotting; D033862:Iron-Binding Proteins; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D013552:Swine; D014168:Transferrin / Q000737:chemistry / Q000235:genetics; D033863:Transferrin-Binding Proteins
D001798:Blood Proteins; D002257:Carbonic Anhydrase Inhibitors; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D008670:Metals; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins; C105555:pICA protein, pig; D002256:Carbonic Anhydrases