Isolation and structural characterization of a cDNA clone encoding rat gastric intrinsic factor

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Intrinsic factor (IF) is crucial for cobalamin (Cbl) absorption in the stomach.
• Understanding IF's structure is key to elucidating its function in Cbl uptake.
• Previous studies focused on IF purification and limited sequencing.

Purpose of the Study:

• To determine the primary amino acid sequence of rat intrinsic factor (IF) using cDNA cloning.
• To investigate the functional properties of the primary translation product of IF.
• To propose a structural model for IF based on sequence homology and functional data.

Main Methods:

• Purification and sequencing of rat intrinsic factor (IF) proteolytic fragments.
• Construction and screening of a rat gastric cDNA library using antibody and oligonucleotide probes.
• Sequencing of an isolated IF cDNA clone to predict the primary amino acid sequence.
• Cell-free translation of IF mRNA and assessment of cobalamin (Cbl)-binding activity.

Main Results:

• An IF cDNA clone was isolated and sequenced, predicting a 421-amino acid coding region and a 22-amino acid signal sequence.
• The primary translation product of IF exhibited cobalamin (Cbl)-binding activity without post-translational modification.
• The N-terminal region of IF showed structural and hydropathic homology to nucleotide-binding domains of oxidoreductases, particularly cytoplasmic malate dehydrogenase.

Conclusions:

• The primary translation product of rat intrinsic factor possesses intrinsic cobalamin (Cbl)-binding activity.
• A structural model for IF is proposed, localizing the Cbl-binding domain to the N-terminal half of the protein.
• Sequence homology suggests a functional link between IF's nucleotide-binding domain and those in oxidoreductases.