Abstract

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.

Highlights

  • Microfibrils of similar morphology are found in nonelastic ated glycoprotein (MAGP) were used to screen a ran- tissues such as periodontal ligament, ocular zonule, and mesdom-primed, bovine nuchal ligament cDNA library in angium of renal glomerulus

  • Identification of MAGP cDNA Clones-Several immunopositiveclones were identified by screeningtherandomprimed cDNA library with affinity-purified anti-MAGP an

  • Sequencing of the cDNA in pBluescript revealed an open reading frame that extended for the full-length of the 303-bp clone and which was in phase with the lac Z gene of the original gtll vector [20]

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Summary

THEJOURNAOLF BIOLOGICACLHEMISTRY

Vol 266, No 12, Issue of April 25, pp. 7596-7601,1991 Printed in U.S.A. Complementary DNA Cloning Establishes Microfibril-associated Glycoprotein (MAGP) to Be a Discrete Component of the Elastinassociated Microfibrils*. Four additional glycoproteins with apparent molecular masses of 340,78,70, and 25 kDa were identified in such extracts of developing bovine indicated that MAGP contains two structuralldyissim- nuchal ligament. It was demonstrated using immunoelectron ilar regions, anamino-terminaldomaincontaining microscopy that the340- and 78-kDa specieswere derived highlevels of glutamine,proline, and acidicamino from the microfibrilsand these were named microfibrillar acids and a carboxyl-terminal domain containing all protein 340 (MP340) and microfibrillar protein 78 (MP78), 13of the cysteine residues anmdost of the basic amino respectively ( 5 ). Construction of cDNALibraries-An oligo(dT)-primed Xgtll cDNA library (3.6 X 10' independent clones) was constructed using poly(A+) RNA from the nuchal ligament of a 15-day-old calf.

RESULTS
Gln Ser Val Ala Ala Ala
Comparison of amino acid composition
Proline Serine
DISCUSSION
Methods
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