Direct detection and quantitative determination of bovine lactoferricin and lactoferrin fragments in human gastric contents by affinity mass spectrometry.

Lactoferricin (Lfcin) is a bioactive fragment of lactoferrin derived from the bactericidal and putative lymphocyte receptor binding domain(s) located within the N-lobe of lactoferrin. Although known to be liberated from at least three species of lactoferrin, conditions leading to Lfcin generation in vivo and factors affecting its distribution are still not known. Recently, we have developed a method of surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry using n-butyl terminal groups for surface-enhanced affinity capture (SEAC) to quantify not only Lfcin generated in vivo but also other lactoferrin fragments. Unlike previous efforts to detect lactoferrin and Lfcin with specific antibodies, the SELDI affinity assay distinguished lactoferrin, lactoferrin fragments, Lfcin and unrelated peptides without their interference with each other. To evaluate Lfcin generation in vivo, the experimental design involved feeding 200 mL of 10 mg/mL (1.22 x 10(-4) mol/L) bovine lactoferrin to an adult. Gastric contents were recovered 10 min after ingestion. Lfcin produced in vivo was directly captured by the SEAC device. The amount of Lfcin in the gastric contents was 16.91 +/- 2.65 micrograms/mL (5.350 +/- 0.838 x 10(-6) mol/L). However, a large proportion of the ingested lactoferrin was not completely digested. Lactoferrin fragments containing the Lfcin region were analyzed by in situ hydrolysis with pepsin after being captured by the SEAC device. As much as 5.740 +/- 0.702 x 10(-5) mol/L of the partially degraded lactoferrin fragments were found to contain the Lfcin region, including peptide domains 17-43, 17-44, 12-44, 9-58, and 16-76 of bovine lactoferrin. These results show that bovine Lfcin can be produced in the human stomach after ingestion of an infant formula supplemented with bovine lactoferrin. It is now important to determine whether Lfcin is generated in the intestinal tract of formula-fed and breast-fed infants, and geriatric patients consuming foods enriched with lactoferrin.

The survival of ingested lactoferrin in the gastrointestinal tract of adult mice.

Lactoferrin is an 80 kDa major protein component of mammalian colostral whey. The antimicrobial active centre of lactoferrin, lactoferricin (Lfcin), may also be an important determinant of the interaction between lactoferrin and specific receptors on lymphocytes. We have documented the survival in vivo of ingested lactoferrin in the gastrointestinal tract of adult mice by surface-enhanced laser desorption/ionization affinity MS. Various kinds of degraded lactoferrin fragments were detected as molecular-ion peaks corresponding to Lfcin after being captured by an affinity capture device, hydrolysis in situ and laser desorption/ionization. No evident molecular-ion peaks of Lfcin were observed upon analysis of faeces from mice fed commercial milk, whereas lactoferrin fragments containing the Lfcin region were detected at concentrations in the order of at least pmol/g in the faeces of mice fed milk enriched with lactoferrin at 40 mg/ml. These results suggest that ingested lactoferrin would survive transit through the gastrointestinal tract as partially degraded forms containing the receptor-binding region(s) as well as the antimicrobial active centre.

Bactericidal domain of lactoferrin: detection, quantitation, and characterization of lactoferricin in serum by SELDI affinity mass spectrometry.

Lactoferricin is a bioactive peptide fragment (3196 Da) derived from lactoferrin (80 kDa) that contains the bactericidal domain and the lymphocyte receptor-binding domain of lactoferrin. Although lactoferricin has been produced from lactoferrin by proteolytic digestion in vitro, its natural occurrence and distribution in vivo are still not clear, in part because of the absence of a suitable detection means. Surface-enhanced laser desorption/ionization (SELDI) was used to detect and characterize lactoferricin by affinity mass spectrometry. Human, porcine, and bovine lactoferricin in unfractionated serum samples were found to bind specifically to ligands presenting a terminal n-butyl group. SELDI was used to detect and quantify each species of lactoferricin in a manner that was independent of the presence of intact lactoferrin, partially degraded lactoferrin, and lactoferrin peptides containing the lactoferricin peptide sequence. The limit of detection of bovine lactofericin in serum was as low as 200 pg/ml. The FKCRRWQWR-homoserine/-homoserine lactone moiety of bovine lactoferricin, which includes the complete antimicrobial center (i.e., RRWQWR), was shown to be responsible for interaction with the n-butyl group. The SELDI procedure defined here is the only molecular recognition tool known to date that is capable of distinguishing the multi-functional lactoferricin domain located within structurally related but distinct forms of lactoferrin and its metabolic fragments. Enabling the direct quantitation of lactoferricin produced in vivo opens new opportunities to evaluate lactoferrin function.

Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin.

The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9+/-2.7 microg/ml (5.4+/-0.8 x 10(-6) mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7+/-0.7 x 10(-5) mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in the case of adults, from lactoferrin secreted into saliva.

Phosphorylation of human progesterone receptor by cyclin-dependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo.

The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.

Cryptic antigenic determinants on the extracellular pyruvate dehydrogenase complex/mimeotope found in primary biliary cirrhosis. A probe by affinity mass spectrometry.

Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

Estrogen regulation of lactoferrin expression in human endometrium.

PROBLEM: Lactoferrin is an iron-binding glycoprotein that has been shown to be overexpressed in human endometrial carcinomas. The purpose of our present study is to investigate the possible role of estradiol in the expression of lactoferrin. METHOD OF STUDY: We investigated 1) serum levels of lactoferrin in five women during normal ovulatory cycles, 2) serum levels of lactoferrin during ten human menopausal gonadotropin induced cycles when estradiol levels are high, and 3) lactoferrin expression in five proliferative and five secretory phase endometrium by immunohistochemical studies. The serum concentrations of lactoferrin were measured by a peroxidase-based enzyme-linked immunosorbent assay. RESULTS: In normal ovulatory cycles, the mean serum lactoferrin concentration during the proliferative phase (0.4013 +/- 0.0242 micrograms/mL) was significantly higher (P < 0.02) than in the secretory phase (0.3468 +/- 0.0209 micrograms/mL). In induced cycles, there was gradual increase in lactoferrin levels with increasing estradiol concentrations. Peak lactoferrin levels in induced cycles (0.7495 +/- 0.1148 micrograms/mL) were significantly higher (P < 0.003) than the midcycle levels (0.423 +/- 0.0424 micrograms/mL) in normal cycles. Immunohistochemical analysis of the endometrium revealed greater expression of lactoferrin in proliferative endometrium (50.7 +/- 13%, range 28-72%) than in secretory endometrium (19.2 +/- 4%, range 7-31%). CONCLUSION: These results indicate that estradiol may play a role in the regulation of lactoferrin expression in human endometrium.

Polymers as surface-based tethers with photolytic triggers enabling laser-induced release/desorption of covalently bound molecules.

A novel design for surface-based macromolecular docking and release is presented together with a strategy to improve and extend biopolymer structure determination capabilities. Polymeric surfaces with arrays of tethers for covalent molecular attachment were designed with photolytic triggers to enable spatially defined, laser-induced uncoupling/desorption of the tethered molecules. Upon photolytic cleavage, a defined portion of the tether ("tail") remains attached to the biomolecule as a probe. Chemically defined memory, determined by the number of reporter tails, reflects the biomolecule interaction with tether-probe devices encountered (i.e., footprint) on the probe surface. To demonstrate function, a surface of poly(4-vinylpyridine) was extended through the pyridinium nitrogens with spacer arms (-N-ethylsuccinamyl-) producing photolytic pyridinium nitrogen bonds. The photolabile tether was terminated with leaving groups (N-hydroxysulfosuccinimide) for covalent attachment of biopolymers. An 18-residue peptide (N terminus of human beta-casein) was covalently docked to these tether-probes, irradiated with coherent UV light, and released with two reporter tails of a mass predicted by tether formation at the two primary amine groups and subsequent photolytic cleavage at the intended site. This is the first demonstration of polymeric surface structure enabling the covalent docking and laser-induced uncoupling/desorption of intact macromolecules through the use of photolytic tethers. Surface-based tether-probe devices, operated by coherent light, should advance our ability to explore covalent modifications in biopolymer structure and alterations in conformation, generated either in advance of tethering or through chemical/enzymatic manipulations performed directly in situ.

Quantitative analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry.

Quantitative aspects of oligonucleotide analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry remain largely unexplored relative to the efforts that have been devoted to quantitative peptide and protein analysis. The successful quantitation of these other biopolymers coupled with the potential of rapid nucleic acid analysis by desorption/ionization techniques prompted the present investigation into quantifying mixed base oligonucleotides of intermediate molecular weights. This report describes the concentration-dependent desorption/ionization of a 21-base oligonucleotide (MW 6361) using a 36-base oligonucleotide (MW 11 131) as an internal standard. Peak height and peak area ratios (analyte to internal standard) varied linearly as a function of oligonucleotide concentration (R2 = 0.966 and 0.991, respectively). The linearity of response extended over nearly three orders of magnitude, from 0.125 to 100 pmol of analyte applied. The use of an internal standard improved the linearity of the calibration curve and reduced relative standard deviations. These results demonstrate for the first time the quantitation of medium size oligonucleotides using MALDI.

Quantitation of metal isotope ratios by laser desorption time-of-flight mass spectrometry.

Laser desorption time-of-flight mass spectrometry (LD/TOF-MS) is evaluated for the determination of stable metal isotope ratios. The isotope ratios of five metal ions (Cu, Ca, Mg, Fe, Zn) in atomic absorption standard solutions and two metal ions (Ca, Mg) in human serum samples are determined. With an existing LD/TOF-MS instrument we show that the technique can overcome the difficulties of the most commonly used methods for measuring metal isotope ratios: (1) all metals are ionizable without surface treatment, thus overcoming the major drawback of thermal ionization mass spectrometry (TIMS); (2) there is no matrix involved to interfere with the metal ion detection, thus overcoming the major disadvantage of inductively coupled plasma mass spectrometry (ICPMS); (3) there is no interference from hydride ions, a major disadvantage of fast atom bombardment secondary ionization mass spectrometry; (4) a mixture of metals can be detected simultaneously using a single laser wavelength, overcoming the major disadvantage of resonance ionization mass spectrometry; (5) accuracy and precision comparable to ICPMS can be achieved with the current instrumentation; (6) precision comparable to TIMS is feasible; and most importantly (7) high precision can be achieved on very small quantities of material because the LD/TOF-MS instrument permits all masses to be monitored simultaneously and very small differences in isotope ratio can be detected.

Receptor-mediated binding of milk lactoferrin to nursing piglet enterocytes: a model for studies on absorption of lactoferrin-bound iron.

Lactoferrin, an iron-binding glycoprotein that is abundant in milk of some species, has been suggested to play a key role in the absorption of iron in human infants. This hypothesis is based on the dominant role of lactoferrin as an iron-binding component in human milk and on the occurrence of lactoferrin receptors in brush-border membranes in infants' intestines. The piglet may be a useful model to evaluate the biological activity of lactoferrin because we have previously found the presence of a lactoferrin receptor in brush-border membranes from suckling piglets. In this study, viable enterocytes were isolated from 6- to 20-day-old suckling piglets. Binding studies were performed at 4 degrees C using 125I-labeled porcine lactoferrin. Scatchard analysis of equilibrium binding data showed an apparent binding constant (Kd) of 2 x 10(-6) M (SD = 0.6 x 10(-6)). This affinity is in close agreement with previous results obtained using isolated brush-border membrane vesicles. Bovine lactoferrin inhibited the binding of porcine lactoferrin. Porcine transferrin, however, did not affect porcine lactoferrin binding significantly. Thus, lactoferrin binding is highly specific. When enterocytes were incubated with 125I-labeled lactoferrin at 37 degrees C, the amount of cell-associated radioactivity exceeded the surface binding capacity of the cells by almost fivefold. This finding agrees with the continuous binding and subsequent internalization of 125I-labeled lactoferrin. The isolated piglet enterocyte seems to provide a useful model for further studies of the mechanism of receptor-mediated absorption of lactoferrin.

Marker proteins in the particulate fraction of third-trimester amniotic fluid.

The present clinical evaluation of fetal lung maturity relies largely on the determination of the amniotic surfactant phospholipids phosphotidylglycerol, lecithin, and sphingomyelin, but there are many false negatives as well as false positives among diabetics. The use of other components of lung surfactant, namely, the hydrophobic surfactant proteins (SPs) has long been suggested as an alternative to the classical assay, but tests based on the detection of immunoreactive SP-A have not proved superior or supplanted phospholipid ratios as an index. This report investigates the proteins in a fraction of third-trimester human amniotic fluid (the particulate fraction) enriched in the SP complexes that form the surfactant monolayer. The proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining and immunoblotting. Eight proteins are of particular interest. Three novel proteins (termed AFPP-1, AFPP-4, and AFPP-8) and the alpha-fetoprotein/human serum albumin complex (AFPP-7) can be detected throughout the 28- to 38-week gestational window. The protein that is referred to as AFPP-2 could be identified as SP-A on the basis of immunologic cross-reactivity as well as size and charge characteristics. The time course of appearance of AFPP-2 was also followed in patients with Rh isoimmunization syndrome and was found to be the same as that seen for SP-A. The SP-A was detected as at least five major charged isoforms with multiple subisoforms of different molecular weight and can be distinguished from a related set of proteins (AFPP-5) that appear with a different time course but are possible precursors. Two other proteins (AFPP-3, AFPP-6), which are detectable inconsistently bear some similarity to others reported previously but not extensively characterized. These results define both constant and variable proteins of the particulate fraction of the amniotic fluid and indicate that certain protein isoforms are changing throughout the third trimester. These data enhance the possibility of the utilization of these proteins as markers of lung maturity in conditions such as maternal diabetes.

Protein requirements: from nitrogen balance to functional impact.

Today because of tools capable of probing the details of metabolism, we are able to study the utilization of amino acids in specific, physiologically important processes. Such information may lead to recommended dietary allowances for each amino acid specific for subpopulations and perhaps individuals. These new findings identify the metabolic end-products of some amino acids as crucial intermediates in the maintenance of a variety of physiological functions that bear no direct relationship to protein metabolism. The precursor amino acids for these intermediate compounds are either non-essential or conditionally essential and thus may play a specific, non-protein, role in amino acid requirements in individuals close to nitrogen equilibrium. Another exciting area arises from the marked dissimilarity between the amino acid composition of milk proteins and other body proteins, suggesting that some milk proteins may have evolved to serve unique functions through specific digestion products. Such products may be important in micronutrient absorption in the neonate. Thus, amino acid requirements appear to reach beyond the tradition areas of nitrogen and protein metabolism.

Immobilized metal ion affinity chromatography.

This article describes the technique of immobilized metal ion affinity chromatography (IMAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.

Proposed mechanisms for the involvement of lactoferrin in the hydrolysis of nucleic acids.

Lactoferrin has recently been proposed to have ribonuclease activity in the absence of bound iron. We and others have demonstrated previously that lactoferrin interacts with DNA and will bind a number of transition metal ions via surface-exposed histidyl residues. In the present study, we investigated the possibility that surface-bound copper ions on lactoferrin may catalyze the production of active oxygen species responsible for the hydrolysis of nucleic acids. Purified lactoferrin (apo- and holo-forms) was incubated with CuCl2 in solution to obtain lactoferrin with surface binding sites saturated by Cu(II)ions. the lactoferrin-Cu(II) complex was purified by Bio-Gel P-6 chromatography columns and tested for hydrolytic activity against DNA and RNA as analyzed by agarose gel electrophoresis. Incubation of lactoferrin-Cu(II) complexes with supercoiled plasmid Bluescript II SK DNA led to the rapid formation of relaxed open circular or linear forms of DNA characterized by changed electrophoretic mobility. Lactoferrin with bound Cu(II) also caused extensive degradation of yeast tRNA molecules in the presence of hydrogen peroxide. Covalent modification of surface-exposed histidyl residues by carboxyethylation with diethylpyrocarbonate abolished the lactoferrin-associated hydrolytic activity. These results indicate that lactoferrin-bound Cu(II) can indeed facilitate the hydrolysis of DNA and RNA molecules. Copper-binding sites on lactoferrin appear to serve as centers for repeated production of hydroxyl radicals via a Fenton-type Haber-Weiss reaction. Enhanced nuclease activity associated with elevated local concentrations of lactoferrin would promote microbial degradation.

Maternal lactoferrin in the urine of preterm infants. Evidence for retention of structure and function.

Intact (i.e., 78-kDa) lactoferrin has been purified from the urine of preterm infants fed human milk. The maternal origin of this lactoferrin, and the integrity of its primary structure have been documented. Computer analyses of the circular dichroism spectra revealed a composite secondary structure for the urinary lactoferrin that was indistinguishable from that of purified human milk lactoferrin and similar to that observed in the crystal structure. Intact function was suggested by iron binding; an approximate 2:1 molar ratio of iron to lactoferrin was confirmed. Thus, maternal lactoferrin is absorbed intact by the preterm infant and appears to remain structurally and functionally intact within the circulatory system and during urinary excretion. It is possible, therefore, that maternal lactoferrin has an immunoregulatory influence in newborn infants fed human milk.

Lactoferrin-receptor interaction. Effect of surface exposed histidine residues.

Specific receptors for lactoferrin (Lf) have been identified in the small intestine brush-border membrane and on lymphocytes. Little is known about the mechanism of Lf interaction with its receptor protein. Because of the pH dependence of this interaction, the possible involvement of surface exposed histidine residues was explored. His residues on purified human milk Lf were progressively modified by carboxyethylation with diethyl pyrocarbonate; the reaction was monitored by UV absorbance and by affinity chromatography on immobilized Cu(II) ions. Human infant brush-border membrane vesicles (BBMVs) were prepared by differential centrifugation and precipitation methods. Human peripheral lymphocytes were prepared by Ficoll centrifugation and stimulated by mitogen (PHA) exposure in culture. A competitive receptor binding assay was performed with 59Fe- and/or 125I-labelled native Lf and His-modified Lf. Binding assays with labelled native Lf were conducted in the presence of a 10-200 fold molar excess of DEPC-Lf, the DEPC-Lf was not competitive. A 100-fold molar excess of native Lf completely blocked the interaction of labelled Lf with the receptor. Similar results were obtained for receptor proteins on BBMVs and lymphocytes. Thus, Lf receptors on these two different cell types appear to require His residues for Lf binding.