Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen- and collagen-like domains.
We have previously identified and purified transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes (Ichijo, H., Rönnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., and Miyazono, K. (1991) J. Biol. Chem. 266, 22459-22464). One of these TGF-beta 1-binding proteins, with a molecular weight of 40,000, was purified to homogeneity and subjected to amino acid sequence analysis. The amino acid sequences obtained were used to isolate two closely related cDNA clones from a porcine uterus cDNA library. The deduced amino acid sequences revealed that both cDNAs encoded proteins that were mainly composed of fibrinogen-like and collagen-like domains. Therefore, they were denoted ficolin-alpha and ficolin-beta. Expression of ficolin-alpha and -beta cDNA in mammalian cells revealed that ficolin forms dimers, trimers, and several higher order of oligomers, whose molecular weights fit well with those of the purified TGF-beta 1-binding proteins from porcine uterus. Moreover, immunoblotting analysis using a peptide anti-serum against ficolin indicated that the TGF-beta 1-binding proteins identified in porcine uterus are ficolin-alpha, -beta, and their oligomers or closely related molecules. However, recombinant ficolin-alpha and -beta did not bind TGF-beta 1, despite the similarities in molecular weights and immunoreactivity with the material from the natural source. It is possible that a specific posttranslational modification of ficolin or interaction with another component is needed for TGF-beta 1 binding. Analysis by Northern blotting revealed that the expression of ficolin-alpha mRNA is relatively restricted and most abundant in placenta and lung. On the other hand, ficolin-beta was mainly expressed in skeletal muscle. The in vivo functions of ficolin will be discussed.
Ichijo
H
H
Ludwig Institute for Cancer Research, Uppsala, Sweden.
Hellman
U
U
Wernstedt
C
C
Gonez
L J
LJ
Claesson-Welsh
L
L
Heldin
C H
CH
Miyazono
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D003094:Collagen / Q000235:genetics*; D004247:DNA; D005260:Female; D005340:Fibrinogen / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D037102:Lectins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014599:Uterus / Q000378:metabolism*; D000097604:Ficolins
D002352:Carrier Proteins; D003094:Collagen; D004247:DNA; D005340:Fibrinogen; D037102:Lectins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011061:Poly A; D012313:RNA; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000097604:Ficolins