A nucleotide insertion and frameshift cause albumin Kenitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges

Abstract

Albumin Ke Ânitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Ke Ânitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Ke Ânitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S±S bridges and showed that albumin Ke Ânitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592±597.

Trypsin-Based Monolithic Bioreactor Coupled On-Line with LC/MS/MS System for Protein Digestion and Variant Identification in Standard Solutions and Serum Samples

“…Human serum albumin was isolated from fresh serum and lyophilized. 35 β-Lactoglobulin genetic variants A and B were purified from bovine milk as previously described. 36 Transthyretin from human plasma, dithiothreitol, guanidine hydrochloride, iodoacetic acid, 2-mercaptoethanol were purchased from Sigma (St. Louis, MO).…”

“…Water was deionized by passing through a Direct-Q (Millipore) system (Millipore, Bedford, MA). Human serum albumin was isolated from fresh serum and lyophilized .…”

Immobilised cooper(II) ion-affinity chromatography of natural mutants of human serum albumin and proalbumin

“…imidazole side chains). The genetic variants of HSA differ from wild-type albumin by only a few changes in the primary structure [6,17]. The question of the number andidentity of the histidines involved has not been answered, however.…”

“…More than 60 structurally different genetic variants of serum albumin and proalbumin have been identified and sequenced at the DNA and/or protein level [6,17]. More than 60 structurally different genetic variants of serum albumin and proalbumin have been identified and sequenced at the DNA and/or protein level [6,17].…”