Cloning and characterization of porcine enamelin mRNAs.
Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.
Hu
C C
CC
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry 78284-7888, USA.
Fukae
M
M
Uchida
T
T
Qian
Q
Q
Zhang
C H
CH
Ryu
O H
OH
Tanabe
T
T
Yamakoshi
Y
Y
Murakami
C
C
Dohi
N
N
Shimizu
M
M
Simmer
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D003743:Dental Enamel / Q000378:metabolism; D003746:Dental Enamel Proteins / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D013552:Swine
D017931:DNA Primers; D003746:Dental Enamel Proteins; D012333:RNA, Messenger; C038456:tuftelin