Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastin-associated microfibrils.
Affinity-purified antibodies to microfibril-associated glycoprotein (MAGP) were used to screen a random-primed, bovine nuchal ligament cDNA library in lambda gt11. A 303-base pair clone, cM5, was isolated which encoded an amino acid sequence homologous with that determined directly from a Lys-C peptide of MAGP. A 936-base pair cDNA clone, cM32, was identified in an oligo(dT)-primed cDNA library using plaque hybridization with clone cM5. Clone cM32 encoded amino acid sequences corresponding to sequences obtained from three Lys-C peptides of MAGP, indicating that the clone was an authentic cDNA for the glycoprotein. The cDNA coded for the entire MAGP polypeptide (21 kDa) of 183 amino acids including a putative signal peptide of 17-19 amino acids. This was confirmed by in vitro translation of synthetic mRNAs transcribed from cM32. The amino acid composition of the encoded protein was virtually identical to that previously published for MAGP. DNA sequence analysis of cM32 indicated that MAGP contains two structurally dissimilar regions, an amino-terminal domain containing high levels of glutamine, proline, and acidic amino acids and a carboxyl-terminal domain containing all 13 of the cysteine residues and most of the basic amino acids. Northern blot hybridization of poly(A+) RNA from fetal nuchal ligament with clone cM32 identified a single mRNA species for MAGP of approximately 1.1 kilobases. The evidence indicates that MAGP is a distinct component of 12-nm microfibrils and that it is not derived from a larger microfibrillar glycopolypeptide.
Gibson
M A
MA
Department of Pathology, University of Adelaide, South Australia.
Sandberg
L B
LB
Grosso
L E
LE
Cleary
E G
EG
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002474:Cell-Free System; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004547:Elastic Tissue / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000737:chemistry; D015183:Restriction Mapping
D000596:Amino Acids; D003285:Contractile Proteins; D016326:Extracellular Matrix Proteins; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; C016598:microfibrillar protein; D004247:DNA