Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.     

Preliminary crystallographic studies on duck ovotransferrin

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.     

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.     

Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila.

The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.     

Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.     

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.     

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.     

Cancer prevention by bovine lactoferrin: from animal studies to human trial

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and, despite improved colonoscopic screening, CRC is a leading cause of death from cancer. Administration of bovine lactoferrin (bLF) suppresses carcinogenesis in the colon and other organs of test animals, and recently it was shown that ingestion of bLF inhibits the growth of adenomatous polyps in human patients. Here we review work which established bLF as an anti-carcinogenic agent in laboratory animals and the results of a clinical trial which demonstrated that bLF can reduce the risk of colon carcinogenesis in humans.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.     

Preliminary crystallographic studies of the Fab fragment of an anti-azophenylarsonate antibody

Single crystals of the Fab fragment of a murine A/J anti-azophenylarsonate monoclonal antibody have been prepared by the vapor diffusion method. Antibody 3A7 uses the same combination of variable region gene segments (VK, JK, VH, JH) as do anti-azophenylarsonate antibodies bearing a predominant cross-reactive idiotype, but utilizes a different D gene segment. The crystals grow in the presence of beta-octylglucoside as tetragonal bipyramids in the space group of either P4(1)2(1)2 or P4(3)3(1)2 and with unit cell dimensions of a = b = 77.9 A, and c = 146.7 A. They diffract X-rays to better than 2.7 A resolution. Data up to 2.7 A resolution have been collected.     

Twenty-five years of research on bovine lactoferrin applications

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.     

Preliminary crystallographic studies of glycosomal glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei

Crystals of glyceraldehyde phosphate dehydrogenase from the glycosome of Trypanosoma brucei brucei have been grown, and a partial data set has been collected using synchrotron radiation. The crystals diffract initially to 2.3 A resolution. The space group is P2(1)2(1)2, with cell dimensions a = 135 A, b = 255 A, c = 115 A, so there are probably at least two tetramers in the asymmetric unit.     

Quality control of commercial bovine lactoferrin

Herein we review commercial bovine lactoferrin quality issues by describing an example of industrial production, the current status of global quality standardization, and quality-activity concerns for further discussion. Morinaga Milk Industry has been industrially producing bovine lactoferrin in Milei GmbH, Germany, since 1989. We delineate its production and quality as an example of safe and high-quality manufacturing. Currently, global standardization in the quality of bovine lactoferrin is progressing through Novel Food and GRAS in the EU and USA, respectively. Novel Food was applied or notified to seven lactoferrin manufacturers and GRAS was notified to three manufacturers, two of which are for infant use and one is for adult use, by the end of 2017. The specifications of these regulations are relatively high, including more than 95% lactoferrin purity in protein, which means that such companies can supply relatively high-grade lactoferrin. There appear to be several concerns regarding lactoferrin quality affecting activities, including contamination of lipopolysaccharide (LPS) and angiogenin, purity, and degradation of lactoferrin sample. Although LPS is immunologically toxic when invading the body, it is distributed normally in foods and the gut. However, an industrial lactoferrin sample may contain LPS at a maximum LPS/lactoferrin molecule ratio?=?1/1724, which means 99.9% of the lactoferrin molecule is LPS-free. It is difficult to speculate that LPS contained in a lactoferrin sample affects its activities. Finally in order to achieve good and reproducible results, we make proposals to researchers a use of high-grade lactoferrin, careful storage, and indication the manufacturers’ names and specifications in the paper.