John W. Tweedie

Three-Dimensional Structure of Lactoferrin

Lactoferrin has many demonstrated activities. Some of these undoubtedly correspond to important in vivo functions; others may only apply in vitro, but may nevertheless lead to possible uses for lactoferrin in medicine or in biotechnology. In either case, the key to understanding the molecular basis of these activities, and ultimately being able to manipulate them, resides in the three-dimensional structure of the protein.

Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following treatment with a restriction endonuclease or DNA topoisomerase I

A two‐session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of DNA with a restriction endonuclease and with a DNA topoisomerase. This session introduces students to the concept of DNA topoisomers, to the properties of different forms of DNA, and to the activity of restriction endonucleases and topoisomerases toward these forms. The exercise also involves measuring the size of linear duplex fragments of DNA by comparison of mobility with a ladder of double stranded DNA of known sizes.

Cloning and Characterisation of the cDNA for Sheep Liver Cytosolic Aldehyde Dehydrogenase

Sheep liver cytosolic aldehyde dehydrogenase (AIDH) has been studied extensively by others from this department (reviewed in Blackwell et al., 1989). In order to extend these studies using site-directed mutagenesis it was necessary first to isolate and sequence the cDNA for this form of the enzyme. The cDNA sequences for the human liver cytosolic (Hsu et al., 1989) and mitochondrial iso-forms of AIDH (Hsu et al., 1988) were used to design a probe which would allow specific identification of cytosolic AIDH. We report here the isolation and sequencing of a full-length clone for sheep liver cytosolic AIDH.

Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.

Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe(3+) ion and an associated CO(3)(2-) ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO(3)(2-) ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129-138], the structure has been refined at 3.0 A resolution to an R factor of 13.9% (R(free) of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54 degrees ) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321-332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.

Cloning and Expression of the C-Terminal Lobe of Human Lactoferrin

Three dimensional studies of human lactoferrin (Anderson et al, 1989) have shown that like all other members of the transferrin family, lactoferrin is divided into two lobes; the N-terminal and the C-terminal lobes. Each lobe is capable of synergistically binding one Fe3+ ion and one anion. The cloning of the cDNA for human lactoferrin (hLf) and its subsequent expression in mammalian cells (Stowell et al, 1991) has provided an excellent system to probe the structure and function of hLf by site-directed mutagenesis. The first mutant to be cloned and expressed using this system was the N-terminal lobe (LfN) of hLf (Day et al, 1992). Recombinant protein concentrations of up to 30 mg/1 in the tissue culture medium have been obtained.

Human Class 1 Aldehyde Dehydrogenase

Human Class 1 and Class 2 aldehyde dehydrogenases have been sequenced at both the protein (Hempel et al., 1984, 1985) and DNA level (Hsu et al., 1988, 1989). Studies on the tertiary structure of aldehyde dehydrogenase are in progress (Baker et al., 1995, sheep Class 1; Hurley and Weiner, 1992, beef Class 2), but are not sufficiently advanced to suggest which amino acid residues are important in catalysis. Cys 302 is the only completely conserved cysteine in all known forms of the enzyme (Hempel et al., 1993), and labelling by various substrates and substrate analogues (von Bahr-Lindstrom et al., 1985; Kitson et al., 1991; Pietruszko et al., 1993) has implicated this residue as the probable active site nucleophile. This has been confirmed for the Class 2 enzyme by site-directed mutagenesis (Weiner et al., 1991). In order to establish whether Cys 302 is also the active site nucleophile for Class 1 aldehyde dehydrogenase we decided to carry out mutagenesis at Cys 302. A separate mutant was constructed in which Cys 301, the adjacent residue, was changed to alanine while Cys 302 was left unchanged.

Altered Domain Closure and Iron Binding in Lactoferrin Mutants

Two features of the functional properties of lactoferrin are its ability to bind iron exceptionally tightly and the coupling of rigid-body domain movements to iron binding and release. The latter cause transitions between open and closed forms of the protein. Using site-directed mutagenesis and X-ray crystallography we have examined the importance of selected residues, including the iron ligands Asp 60 and His 253, the anion-binding Arg 121, and Pro 251 in the hinge region. Five mutants, D60S, R121S, R121E, H253M, and P251 A, have been prepared in the context of the N-terminal half-molecule of human lactoferrin, Lfn, and three-dimensional structures have been determined in each case. In D60S the mutation leads to weakened iron binding because a water molecule binds to the iron atom in place of Asp 60. Interdomain interactions are also weakened, and the loss of the Asp side-chain causes a significant change in domain closure; the domains move closer together by 7° in the mutant. The R121S and R121E mutants show altered anion binding and very small changes in domain orientations. The H253M and P251A mutants show identical domain closure to wild-type LfN, but the iron site is altered in H253M; the Met 253 side-chain is not bound to iron, leaving a 5-coordinate site. These results are interpreted in terms of the roles of each of the residues in iron binding and release.

Mutagenesis of Human Lactoferrin and Expression in Baby Hamster Kidney Cells

We have previously reported the expression of both full-length recombinant lactoferrin and the recombinant N-lobe half-molecule in baby hamster kidney (BHK) cells (Stowell et al, 1991; Day et al., 1992). The properties of the full-length recombinant protein produced in this system were virtually indistinguishable from those of the native protein isolated from human milk, except for an increased resistance of a minor fraction of the protein to deglycosylation by PNGase. The N-lobe recombinant protein has been characterized (Day et al., 1992) and the structure determined by X-ray crystallography (Day et al., 1993).

Recombinant Human Lactoferrin and Its Variants

Human lactoferrin (lactoferrin) has been shown to bind to receptors present in the human small intestine and on various other types of cells. Little is known about the structural features of the lactoferrin molecule that are needed for receptor recognition. The lactoferrin gene has been cloned and sequenced and recombinant lactoferrin has been expressed in baby hamster kidney cells. The recombinant lactoferrin has been shown to have normal iron-binding properties, but glycosylation of the recombinant lactoferrin appears to differ from that of the native lactoferrin. This expression system has also made it possible to use site-directed mutagenesis to produce variants of human lactoferrin. In this study, we analyze the physical characteristics as well as the receptor binding properties of recombinant lactoferrin and its structural variants, including the N-lobe, N-lobe treated with PNGase, and the N-lobe with its glycosylation site (N137A) mutated. Laser-induced desorption/ionization time-of-flight mass spectrometry is used to evaluate differences between the predicted and observed molecular mass values as a function of posttranslational modification. Competitive binding experiments are conducted with both native and recombinant human lactoferrin to assess receptor binding properties using human brush-border membranes. Each of the recombinant lactoferrin proteins competes effectively with native lactoferrin for receptor binding. The N-lobe alone appears to have a greater affinity for the binding sites than does the intact native lactoferrin. We conclude from these studies that the presence of glycans is not essential for receptor recognition and that the N-lobe of lactoferrin is both necessary and probably sufficient to allow normal binding to the receptor(s).