Maria Cavaletto

Milk Fat Globule Membrane Components–A Proteomic Approach

The milk fat globule membrane (MFGM) is the membrane surrounding lipid droplets during their secretion in the alveolar lumen of the lactating mammary gland. MFGM proteins represent only 1-4% of total milk protein content; nevertheless, the MFGM consists of a complex system of integral and peripheral proteins, enzymes, and lipids. Despite their low classical nutritional value, MFGM proteins have been reported to play an important role in various cellular processes and defense mechanisms in the newborn. Using a proteomic approach, such as high-resolution, two-dimensional electrophoresis followed by direct protein identification by mass spectrometry, it has been possible to comprehensively characterize the subcellular organization of MFGM. This chapter covers the description of MFGM proteomics from the first studies about 10 years ago through the most recent papers. Most of the investigations deal with MFGMs from human and cow milk.

Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination.

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down‐regulation of oxidative damage‐related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae‐ and Gi. margarita‐colonised plants. Up‐regulation of multiple forms of glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae‐inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up‐regulated protein by arsenic treatment.

Human proteome enhancement: High-recovery method and improved two-dimensional map of colostral fat globule membrane proteins

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2 – 4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two‐dimensional electrophoresis (2‐DE) map of the human milk fat globule membrane proteins, both integral and membrane‐associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3 – 10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on 11.5% T homogeneous gels. A reproducible 2‐DE map of integral and membrane‐associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis and/or by amino acid sequencing.

Effects of heavy metals and arbuscular mycorrhiza on the leaf proteome of a selected poplar clone: a time course analysis.

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein.

Proteomic analysis as a tool for investigating arsenic stress in Pteris vittata roots colonized or not by arbuscular mycorrhizal symbiosis.

Pteris vittata can tolerate very high soil arsenic concentration and rapidly accumulates the metalloid in its fronds. However, its tolerance to arsenic has not been completely explored. Arbuscular mycorrhizal (AM) fungi colonize the root of most terrestrial plants, including ferns. Mycorrhizae are known to affect plant responses in many ways: improving plant nutrition, promoting plant tolerance or resistance to pathogens, drought, salinity and heavy metal stresses. It has been observed that plants growing on arsenic polluted soils are usually mycorrhizal and that AM fungi enhance arsenic tolerance in a number of plant species. The aim of the present work was to study the effects of the AM fungus Glomus mosseae on P. vittata plants treated with arsenic using a proteomic approach. Image analysis showed that 37 spots were differently affected (21 identified). Arsenic treatment affected the expression of 14 spots (12 up-regulated and 2 down-regulated), while in presence of G. mosseae modulated 3 spots (1 up-regulated and 2 down-regulated). G. mosseae, in absence of arsenic, modulated 17 spots (13 up-regulated and 4 down-regulated). Arsenic stress was observed even in an arsenic tolerant plant as P. vittata and a protective effect of AM symbiosis toward arsenic stress was observed.

A proteomic approach to evaluate the butyrophilin gene family expression in human milk fat globule membrane.

Human butyrophilin (BTN) expression in milk fat globule (MFGM) was evaluated using two dimensional electrophoresis (2‐DE) as the separation technique, and peptide mass mapping by matrix‐assisted laser desorption/ionisation‐mass spectrometry (MALDI‐MS) as the identification tool. Since milk composition changes throughout lactation time, 2‐DE maps in the pH range 4–7 of colostral MFGM and mature MFGM were compared, showing only slight differences in BTN spot distribution. The BTN gene family codes for seven proteins (BTN, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3), their presence in human tissues has to date been evaluated only at a transcriptional level. Among 70 spots, analyzed and identified by MALDI‐MS, 13 spots were identified as BTN spots and only one as a fragment of BTN2A1. BTN was present in multiple glycoforms, and two smaller BTN forms of about 45 kDa were also identified. We propose an array of BTNs on human MFGM, which could provide breast‐fed infants with immune molecules during the neonatal period.

Isolation and characterization of full and truncated forms of human breast carcinoma protein BA46 from human milk fat globule membranes

We have isolated and characterized two proteins of 50 and 30 kDa from human milk fat globule membranes of healthy donors. N-terminal and internal sequencing revealed that the 50-kDa protein is the full-length human breast carcinoma protein BA46 that is highly expressed in human breast tumors. The 30-kDa protein is a truncated form of protein BA46 which consists of the C-terminal factor V/VIII-like domain of BA46 and which appears to anchor BA46 to the milk fat globule membrane. Defective release of the epidermal growth factor domain containing a surface RGD motif may be related to involvement of BA46 in breast cancer

The Unusual Amino Acid Triplet Asn–Ile–Cys Is a Glycosylation Consensus Site in Human α-Lactalbumin

Human α-lactalbumin has not been described as a glycoprotein, despite the fact that several α-lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the 45Asn in the usual triplet 45Asn–Gly/Gln–47Ser. We have found that human α-lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at 71Asn in the triplet 71Asn–Ile–73Cys, which is conserved in all known α-lactalbumins except red-necked wallaby. That a relatively small proportion of the protien is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of α-lactalbumin.

Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis)

Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds. In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precursor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitellogenin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electrophoretic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable.

Effects of Mercury on Dictyostelium discoideum: Proteomics Reveals the Molecular Mechanisms of Physiological Adaptation and Toxicity

Dictyostelium discoideum amoebae were exposed to Hg 2 microM corresponding to a sublethal concentration and Hg 10 microM with the first effects on mortality and replication rate. A total of 900 spots were visualized by 2-DE electrophoresis. Two-hundred fifty single proteins were identified by mass spectrometry. Low Hg concentration (2 microM) treatment induced up-regulation of 13 spots, mainly involved in oxidative stress response/detoxification, oxidoreductase activity, and metabolic processes. High Hg concentration (10 microM) treatment showed a different PES with 12 proteins downregulated and only two up-regulated, mainly involved in cellular metabolic processes, metal ion binding, and transferase activity. The analyses for the carbonylation show no changes after 2 microM Hg(2+) treatment and 13 differentially carbonylated proteins after 10 microM Hg(2+) involved in a broad range of cellular processes. Our findings provide insight into the mechanisms of physiological adaptation and toxicity to a low and an high mercury concentration, respectively, of Dictyostelium amoebae.