Structural Characterization of Four Genetic Variants of Human Serum Albumin Associated with Alloalbuminemia in Italy

Abstract

A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations : albumins Tregasio, Va1122-Glu, Bergamo, Asp314+Gly, and Maddaloni, Va1.533-Met. The fourth, albumin Besana Brianza, has the same Asp494-Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.Keywords: human serum albumin; genetic variant; amino acid sequence; mass spectrometry; glycosylated alloalbumin.Alloalbuminemia is an inherited condition, which is clinically harmless, caused by a structural variant of human serum albumin. More than 100 allotypes have been found worldwide during screening of plasma proteins by routine clinical electrophoresis or in population genetics surveys with a frequency in the average population of 3.0X10-4-1.0X10~3 [I]. Many of the allotypes have been studied structurally as markers of neutral molecular evolution and because of interest in their frequency, population distribution, and ligand-binding properties. A systematic study carried out in several laboratories by protein and/ or DNA sequencing has so far identified in the albumin gene 49 single-point changes, four chain-termination mutants, and five defects causing analbuminemia (see Table 4 this involves a CpG dinucleotide in the gene and gives rise to a glycosylation site. The variant bears the same complex biantennary structure found in two other glycosylated alloalbumins, albumins Casebrook [I61 and Dalakarlia [17], but also a minor monosialylated form is present. In contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated, the ratio between the glycosylated and unglycosylated forms being about 4: 1. MATERIALS AND METHODSElectrophoretic survey. Fresh specimens of serum from individuals with albumin Bergamo, Besana Brianza, Maddaloni, and Tregasio traits were supplied by Roberto Cesati. Each variant was named according to its geographical origin. All the donors were heterozygous and inheritance of the traits had been demonstated. A preliminary screening of the variants was per-

Human serum albumin: From bench to bedside

“…To date, three HSA N-glycosylated variants have been identified, namely Alb Dalakarlia-1 (glycosylated at Asp63) (Arai et al, 1990;Carlson et al, 1992;Sakamoto et al, 1995), Alb Redhill (glycosylated at Asn318) (Brennan et al, 1990a), and Alb Casebrook (glycosylated at Asp494) (Peach and Brennan, 1991;Haynes et al, 1992;Minchiotti et al, 1997;Nielsen et al, 1997) (Table 1).…”

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

“…These studies have allowed the characterization by protein and/or DNA sequence analysis of more than 60 different genetic mutations [6,7] (see the continuously updated website at http://www.albumin.org) and have provided a unique insight into albumin's ligand binding sites [6,8,9]. Nearly all the known albumin mutations occur on the molecular surface and most of them are clustered in three regions: the propeptide and N‐terminal region, and the C‐terminal parts of subdomains IIB and IIIB [10]. The vast majority of them reflects the existence of single‐base changes in the structural gene, and the recurring alloalbumins are associated with mutations in hypermutable CpG dinucleotides [11,12].…”

“…Normal HSA is not glycosylated but there are three point mutations that give rise to the canonical Asn‐Xaa‐Thr/Ser tripeptide acceptor sequence for N‐glycosylation. In every case, the mutated sequences turned out to be N‐glycosylated: Asp63→Asn [18], Ala320→Thr [19] and Asp494→Asn [10,20].…”

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

“…These studies have allowed the characterization by protein and/or DNA sequence analysis of more than 60 different genetic mutations [6,7] (see the continuously updated website at http://www.albumin.org) and have provided a unique insight into albumin's ligand binding sites [6,8,9]. Nearly all the known albumin mutations occur on the molecular surface and most of them are clustered in three regions: the propeptide and N-terminal region, and the C-terminal parts of subdomains IIB and IIIB [10]. The vast majority of them reflects the existence of single-base changes in the structural gene, and the recurring alloalbumins are associated with mutations in hypermutable CpG dinucleotides [11,12].…”

“…Normal HSA is not glycosylated but there are three point mutations that give rise to the canonical Asn-Xaa-Thr/Ser tripeptide acceptor sequence for N-glycosylation. In every case, the mutated sequences turned out to be N-glycosylated: Asp633Asn [18], Ala3203Thr [19] and Asp4943Asn [10,20].…”