The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF

L.H Tang,H Chokshi,C.B Hu,M.M Gordon,D.H Alpers

doi:10.1016/s0021-9258(18)50044-2

Abstract

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

Highlights

In this report we show that the receptor binding activity is located in the amino-terminal quarter of Intrinsic Factor (IF), whereas all or most of the correctly folded molecule is needed for Cbl binding
The present work begins the identification of the binding sites on intrinsic factor (IF) for cobalamin (Cbl) and the IF
This analysis is consistent with the need for extensive folding asa requisite for Cbl binding

Summary

RESULTS

Both rat and body recognition was determined following immunoprecipitation of human IF cDNAs were used to take advantage of a wider transfected COS-1 cell medium, by identification of the appropriate range of unique restriction sitesin these cDNAs and truncated sized moleculeby autoradiography of SDS-polyacrylamidegels. The full-length rat (399 residues) or human IF (399 residues) constructs produced an increase in the COS cell media in [“CoICbl binding as determined by the charcoal assay. Construct IFsource Restriction Amino acid Cbl Antibody reconmo.itiobnindine residues site though the rat IF construct containing 303 amino acids out of 399 is made in abundance and can be immunoprecipitated from the culture medium comparably to the full-length IF,

HhdlII

Findings

DISCUSSION

Summary