Barbara H. Bowman

Haptoglobin: The Evolutionary Product of Duplication, Unequal Crossing Over, and Point Mutation

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.

Oyster Ciliary Inhibition by Cystic Fibrosis Factor

Ciliary inhibition in oysters serves as an assay in identifying a serum factor in cystic fibrosis patients and heterozygotes. Seriums from 47 patients With cystic fibrosis antd 19 heterozygotes caused ciliary cessation within 35 minutes, whereas serums from only 2 of 64 individuals without cystic fibrosis inhibited ciliary activity within this time.

Cystic Fibrosis: Characterization of the Inhibitor to Ciliary Action in Oyster Gills

The inhibitor to oyster ciliary activity was isolated from serumof cystic fibrosis patients and heterozygotes.The inhibitinig protein fraction wast a cation as judged by electrophoresis; it had a molecuer weight of 125,000 to 200,000 as judged by gel filtration; antd oil diethylaminoethyl-cellulose chromatography it eluted with the immunoglobulin G fraction. The analogous fraction in normal individuals did not inhibit oyster cillary activity.

Evidence of homology between the β-chain of human haptoglobin and the chymotrypsin family of serine proteases

Amino acid sequences from the β-chain of human haptoglobin are compared with those sequences known for the serine proteases of the chymotrypsin family. In a comparison of some 171 residues of the haptoglobin β-chain (approximately 60% of the protein molecule), approximately 30% of these are identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsin A, bovine chymotrypsin B, porcine elastase, or bovine thrombin B-chain, and an additional 10% are chemically similar. A combined comparison of the haptoglobin β-chain with the above five serine proteases gave an identity of 56% and a chemical similarity of 11%. Similarity of primary structure is also striking around two of the five half-cystinyl residues so far characterized in long lengths of sequence. These data provide substantial evidence that the β-chain of haptoglobin is homologous to the chymotrypsin family of serine proteases. Proposals are also presented to explain the occurrence of internal homology in the N-terminal region of the β-chain.

Serum concentrations of vitamin D-binding protein (Group-specific component) in cystic fibrosis

SummaryVitamin D-binding protein (DBP) concentrations were determined in the sera of 90 cystic fibrosis homozygotes, 57 obligate heterozygotes, and 46 normal controls. Very significantly lower mean concentrations were found in the sera of CF homozygotes compared with both heterozygotes and controls (P<0.01, Wilcoxon Rank Sums Test). Subdivision of the samples by Gc phenotype showed that this relationship held true both in the Gc1 and Gc2-1 phenotypes. The small sample size of the Gc2 genotype makes the significance levels of limited usefulness, but the pattern of variation of DBP levels among CF homozygotes, heterozygotes, and controls was consistent with that observed for the Gc1 and Gc2-1 classes. Haptoglobin levels showed high coefficients of variation when compared among CF homozygotes, obligate heterozygotes, and controls, presumably because of nonspecific elevation in the acute-phase response. Alpha2-macroglobulin levels were, if anything, slightly elevated in CF homozygotes compared with controls, while albumin levels showed no significant mean differences between these groups. Since the DBP concentration does not vary with age nor with levels of vitamin D and its metabolites, we interpret our results to mean that DBP levels are specifically decreased in cystic fibrosis, perhaps as the result of impaired glycosylation of the protein.

Cystic fibrosis: The ciliary inhibitor is a small polypeptide associated with immunoglobulin G

Abstract A small polypeptide within the molecular weight range of 6,000 to 11,000 was found in immunoglobulin fractions from sera of cystic fibrosis homozygotes and heterozygotes. This factor appears to be responsible for interfering with ciliary activity in oyster gills. Since it can be dissociated from IgG without reductive cleavage it must be bound in a non-covalent manner. After its dissociation the IgG fractions from cystic fibrosis sera no longer inhibited ciliary activity. This finding explains the differences previously observed in the molecular weights of the ciliary inhibitor synthesized by cultured cells and that of sera from cystic fibrosis genotypes.

Canine haptoglobin: A unique haptoglobin subunit arrangement

1. Isolated canine haptoglobin behaved identically to the alpha 2 beta 2 structure typical of human haptoglobin type 1-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptoglobin dissociated into alpha beta subunits that separated into alpha and beta chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine alpha chain when compared to human alpha 1 chain. 4. These results provide evidence that there is no inter alpha chain disulphide in canine haptoglobin comparable to the alpha 1 20-alpha 1 20 disulphide in human haptoglobin that links the two alpha beta subunits.

A Transferrin Variant in a Kindred with Cystic Fibrosis

An electrophoretically fast transferrin (Tf) variant, TfB, was found in a cystic fibrosis homozygote. Since neither cystic fibrosis nor the transferrin structural gene has been mapped on human chromosomes, a study was made of this kindred. Genetic markers including ABO, MN, Rh, Fy blood groups and Tf, group-specific component, and haptoglobin serum protein polymorphisms were determined in available members of the kindred. The genes for cystic fibrosis and Tf appear to segregate independently in the kindred, although crossing-over between linked genes in the homozygous cystic fibrosis brother of the propositus could account for the genotypes observed.

Cystic fibrosis: studies with the oyster ciliary assay.

Bioassays using ciliary systems have detected a factor or factors in cystic fibrosis (CF) sera and tissue culture medium derived from CF cells. The typical shortcomings of an assay measuring biological activity have been studied, and the means to overcome the weaknesses of the oyster gill cilia assay have been established. The presence of the cystic fibrosis mucociliary inhibitor (CFMI) in experimental fractions may be determined by accepting data from only those assays in which authentic CF and normal (non‐CF) fractions give defined reactions, by measuring the reaction of each sample at least three times, and by examining each experimental sample at a protein concentration greater than the minimum established in this study. The relative concentrations of the CFMI present in the first steps of purification of serum and medium have been calculated in terms of units of inhibition. Generally, the units of inhibition present in serum and medium fractions from heterozygotes are close to one‐half of that in fractions from homozygous sources. Analogous fractions concentrated from a normal (non‐CF) source never inhibited mucociliary activity, even when tested at nearly 100 times the CF concentration. Ciliary assays utilizing oyster gills are essential for monitoring fractionation procedures aimed at purifying the CFMI, and have been shown to be capable and reliable enough to do so.

A new ceruloplasmin variant, Cp Galveston

An undescribed fast‐migrating serum ceruloplasmin variant has been detected by acrylamide gel clectrophoresis with a double stain technique for ceruloplasmin and haptoglobin. The serum type migrated identically to ascites fluid ceruloplasmin from one alcoholic cirrhosis patient and three ovarian carcinoma patients.