Unique albumin with two silent substitutions (540Thr→Ala and 546Ala→Ser): Insights into how albumin is recycled.

OBJECTIVES

To determine the cause of an albumin abnormality detected by chance on electrospray time-of-flight mass spectrometry (TOF MS) of whole plasma, and to assess its physiological consequences.

METHOD

Plasma was examined by TOF MS and tryptic mapping was used to locate mutation sites and determine the relative expression level of the variant and normal albumins. DNA sequencing was used to precisely define mutations.

RESULTS

Whole protein electrospray TOF MS indicated a decrease of 14Da in the mass of albumin. Peptide mass mapping and DNA sequencing established the presence of two novel heterozygous point mutations (540Thr→Ala and 546Ala→Ser) whose combined mass changes (-30 and +16Da) indicated both mutations occurred on the same allele. Peptide ratios showed the variant albumin was present at a lower level than normal with an expression ratio of approximately 1:2 (variant:normal). Phylogenetic sequence alignments show Thr540 is highly conserved while Ala546 has wide species variation, suggesting 540Thr→Ala might compromise the protein.

CONCLUSION

Both mutations occur close together in domain IIIB, a region involved in albumin scavenging and recycling. In particular, Thr540 is close to His535, a residue directly involved in pH-dependent binding and release of albumin from its recycling neonatal Fc receptor. Compromised receptor binding would explain the low albumin (34g/l) concentration and the diminished variant expression level.