Preferred sites of glycosylphosphatidylinositol modification in folate receptors and constraints in the primary structure of the hydrophobic portion of the signal.
The divergent carboxyl-terminal signal peptides for glycosylphosphatidylinositol (GPI) membrane anchor attachment in folate receptor (FR) types alpha and beta were characterized. All of the candidate amino acid residues for GPI modification were identified and tested by substituting individually and in combination with amino acids that cannot be modified by GPI. Thus the GPI modification in FR-alpha was decreased to 22% by mutation of Ser234 to Thr but unaltered by changing the other candidate, Gly235, to Met. However, the double mutant FR-alpha Ser234-Thr,Gly235-Met showed half of the GPI modification seen in FR-alpha Ser234-Thr. This result suggests that Ser234 is the preferred GPI modification site, while Gly235 is a minor, alternate GPI modification site. Similarly, in FR-beta, mutation of Asn230 to Gln decreased GPI modification to 32%, while mutation of the other candidate site, Gly237, to Met had no effect. However, mutation at both sites further reduced the GPI modification by a half. A five amino acid carboxyl-terminal deletion (FR-beta delta 5) caused no decrease in the extent of GPI modification. However, the same deletion in FR beta Asn230-Gln decreased the residual GPI modification by 66%. These results suggest that Asn230 is the preferred GPI modification site in FR-beta, while Gly235 offers a minor alternate modification site; consistent with this conclusion is the fact that modification at the downstream site is hindered by its proximity to the carboxyl terminus in FR-beta delta 5. Further, the suggestion that the hydrophobic portion of the GPI signal is a random sequence of neutral amino acids with overall moderate hydrophobicity was tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Yan
W
W
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008, USA.
Ratnam
M
M
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011956:Receptors, Cell Surface; D017421:Sequence Analysis
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D009841:Oligonucleotides; D011956:Receptors, Cell Surface