The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 A. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.

Atomic structure and chemistry of human serum albumin.

Publisher Summary This chapter provides an insight of the findings of past significant papers with the current knowledge of the recently determined high resolution X-ray structure of serum albumin. The most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands. The sequences of all albumins are characterized by a unique arrangement of disulfide double loops that repeat as a series of triplets. Albumin belongs to a multigene family of proteins that includes α- fetoprotein (AFP) and vitamin D-binding protein (VDP), also known as G complement (Gc) protein. Although AFP is considered the fetal counterpart of albumin, its binding properties are distinct and it is suggested that AFP may have a higher affinity for some unknown ligands important for fetal development. Domain structure and the arrangement of the disulfides, the surface charge distribution, and the conformational flexibility of the albumin molecule are described. The nature of ligand binding, including small organics, long-chain fatty acids, and metals, to multiple sites on the albumin molecule is clearly depicted. The chapter concludes with the perceptive comments on future directions being taken to explore the structure and function of this fascinating protein.

Structure of serum albumin.

Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the beta subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase Covalent cross-linking of 125I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF

https://science.sciencemag.org/content/sci/251/4995/802.full.pdf

Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.

Molecular cloning and expression of human hepatocyte growth factor

Human serum albumin: from bench to bedside.

The question of how genetic material controls phenotypic expression was posed 60 years ago by Hermann Muller (1922), but the ability to answer the question awaited the development of biochemical techniques capable of dissecting and comparing purified proteins (Moore et al., 1958; Edman and Sjoquist, 1956; Ingram, 1957). Human haptoglobin has served as an unexpected source of information relating the mechanisms involved in genetic rearrangements to the linear sequence of amino acids within a protein. After developing a potent technique, starch gel electrophoresis, which proved capable of resolving and identifying previously unknown inherited variations in many human plasma proteins, Oliver Smithies and co-workers (Smithies, 1959; Smithies et al., 1962a, 1962b) demonstrated that serum haptoglobin molecules from different individuals varied one from another in a heritable way. Haptoglobin has served as an impressive paradigm of how genetic rearrangements can construct a polymorphic system and how, in the evolutionary process, old proteins can obtain new functions by utilizing amino acid sequences which have been ever so slightly altered. Excellent reviews have been written about haptoglobin, which can be consulted for further detail (Javid, 1978; Putnam, 1975; Pintera, 1971; Sutton, 1970; Giblett, 1969; Kirk, 1968; Schultze and Heremans, 1966). This review will emphasize recent developments in analyzing the composition of the haptoglobin molecules and the evolutionary implications of these findings.

Haptoglobin: the evolutionary product of duplication, unequal crossing over, and point mutation.

Transferrin (Tf) is the major iron binding protein in vertebrate serum. It shares homologous amino acid sequences with four other proteins: lactotransferrin, ovotransferrin, melanoma antigen p97, and HuBlym-1. Antigen p97 and the Tf receptor genes have been mapped on human chromosome 3. The goal of the study described here was to initiate the characterization of the Tf gene by identifying and characterizing its cDNA and mapping its chromosomal location. Recombinant plasmids containing human cDNA encoding Tf have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Within the 2.3 kilobase pairs of Tf cDNA analyzed, there is a probable leader sequence encoded by 57 nucleotides followed by 2037 nucleotides that encode the homologous amino and carboxyl domains. During evolution, three areas of the homologous amino and carboxyl domains have been strongly conserved, possibly reflecting functional constraints associated with iron binding. Chromosomal mapping by in situ hybridization and somatic cell hybrid analysis indicate that the Tf gene is located at q21-25 on human chromosome 3, consistent with linkage of the Tf, Tf receptor, and melanoma p97 loci.

https://www.pnas.org/content/pnas/81/9/2752.full.pdf

Human transferrin: cDNA characterization and chromosomal localization

The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions.

https://www.pnas.org/content/pnas/77/6/3388.full.pdf

Covalent structure of human haptoglobin: a serine protease homolog

DNA sequencing shows that the intragenic duplication within the human haptoglobin Hp2 allele was formed by a non-homologous, probably random, crossing-over within different introns of two Hp1 genes, probably in an Hp1F / Hp1S heterozygote.

Duplication within the haptoglobin Hp2 gene.

The group-specific component (Gc) is the major vitamin D-binding protein in plasma. The gene encoding Gc is linked, on human chromosome 4, to the albumin and alpha-fetoprotein genes. These two genes previously were shown to have evolved from a smaller ancestral gene by intragenic triplication. Recombinant plasmids containing human cDNA encoding Gc have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Characterization of Gc cDNA has provided the complete amino acid sequence of the protein and revealed strong sequence homology with albumin and alpha-fetoprotein. Of particular interest is a conserved pattern of disulfide bridges that form the triple-domain structures in albumin, alpha-fetoprotein, and Gc. Gc cDNA was used as a probe in Southern blot analysis of somatic-cell hybrids to confirm that the Gc locus is on chromosome 4.

https://www.pnas.org/content/pnas/82/23/7994.full.pdf

Barbara H. Bowman

Papers

Haptoglobin: the evolutionary product of duplication, unequal crossing over, and point mutation.

Human transferrin: cDNA characterization and chromosomal localization

Covalent structure of human haptoglobin: a serine protease homolog

Duplication within the haptoglobin Hp2 gene.

Human group-specific component (Gc) is a member of the albumin family