Alessandra Olianas

Trafficking and Postsecretory Events Responsible for the Formation of Secreted Human Salivary Peptides A Proteomics Approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and “S type” Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β4 and β10, antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Age-dependent modifications of the human salivary secretory protein complex

Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups.

A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization

We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH(2)-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8°C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.

Chrono-Proteomics of Human Saliva: Variations of the Salivary Proteome during Human Development

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Top-down analytical platforms for the characterization of the human salivary proteome

Comprehensive analysis and characterization of the human salivary proteome is an important step towards the possible use of saliva for diagnostic and prognostic purposes. The contribution of the different sources to whole saliva, and the evaluation of individual variability and physiological modifications have been investigated by top-down proteomic approaches, disclosing the faceted and complex profile of the human salivary proteome. All this information is essential to develop saliva protein biomarkers. In this Review the major results obtained in the field by top-down platforms, and the improvements required to allow a more complete picture, will be discussed.

The intriguing heterogeneity of human salivary proline-rich proteins: Short title: Salivary proline-rich protein species.

UNLABELLED The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood. SIGNIFICANCE The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals.

Modifications of the acidic soluble salivary proteome in human children from birth to the age of 48months investigated by a top-down HPLC-ESI-MS platform.

UNLABELLED During the first year of life the infant oral environment undergoes dramatic changes. To investigate how the salivary proteome of human children evolves during infant development we have analyzed whole saliva of 88 children aged between 0 and 48months by a top-down platform based on RP-HPLC-ESI-MS. Children were divided according to their age into five groups (A, 0-6months, N=17; B, 7-12months, N=14; C, 13-24months, N=32; D, 25-36months, N=16; E, 37-48months, N=9). The proteins and peptides analyzed were histatins (histatin-1, histatin-3 1/24), acidic proline-rich proteins, statherin, P-B peptide, and salivary cystatins. Protein and peptide quantification based on the area of the RP-HPLC-ESI-MS extracted ion current peak evidenced that: (i) concentrations of the major salivary proteins/peptides showed a minimum in the 0-6-month-old group and increased with age; (ii) the level of histatin-1 reached a maximum in the 7-12-month-old group, a minimum in the 13-24-month-aged babies and it increased again in the 25-36-month-old group; (iii) S-type cystatins were almost undetectable in the 0-6-month-old group; (iv) P-B peptide concentration greatly increased with age; (v) histatin-3 1/24 and statherin concentrations did not show any age-related variation. BIOLOGICAL SIGNIFICANCE The top-down proteomic approach undertaken in this work reveals that the salivary proteome of human children from birth to 48months of age shows important quantitative modifications. The concentrations of the major salivary proteins, with the exception of statherin and histatin-3 1/24, showed a minimum in the 0-6-month-old group when the expression in salivary glands is probably not fully activated. Concentrations of the salivary proteins slowly increased with age, with different trends. Only histatin-1 showed the highest concentration in the 7-12-month-old group, followed by a decrease in the 13-24-month-aged children. This particular trend could be related to the phenomenon of eruption of primary dentition. This study gives a contribution to the knowledge on the physiological variability occurring in human saliva during the early childhood. It could represent a strong and reliable basis for further investigation of saliva to develop diagnostic and prognostic biomarkers.

Tyrosinase activity and hemocyanin in the hemolymph of the slipper lobster Scyllarides latus

The respiratory protein hemocyanin is present in molluscans and in some species of arthropods, and its dioxygen binding site strongly resembles that of the monophenol-hydroxylating and catechol-quinonising enzyme tyrosinase. Moreover, some hemocyanins show a certain extent of tyrosinase activity, so a common ancestry between the two proteins has been suggested. However, in the case purified hemocyanin of Scyllarides latus any attempts to evoke tyrosinase activity failed. A distinct tyrosinase has been purified to homogeneity from the hemolymph, and kinetically characterised. The purified tyrosinase showed both monophenolase and diphenolase enzyme activity and therefore it could be well defined as a true tyrosinase. This finding suggests that in the case of the studied crustacean the evolutionary functional divergence between dioxygen transport and oxidation of phenolics has already reached its completeness.

Benthic mucilagenous aggregates: Biochemical characterization and ligand binding properties

Abstract The biochemical composition of benthic mucilaginous aggregates collected from the south-western coastal waters of Sardinia in the Mediterranean Sea was analysed. Acidic glycoproteins surrounding the included microorganisms, mainly diatoms, were found to be the predominant fraction. The aggregates, diluted and finely dispersed by ultrasonic treatment, bound with ammoniated ruthenium oxychloride (ruthenium red), a specific ligand for the acidic glycoproteins. When concentrated crude aggregates were used, ruthenium red was bound both chemically and by physical entrapment. The data were consistent with the Langmuir ligand binding isotherm. The extent of physical adsorption displayed a clear pH dependence. Physical adsorption was predominant at acidic pH, suggesting that the stabilization of the supramolecular structure of the aggregates also depends on the degree of protonation of the acidic glycoresidues. A structural model of the aggregates is proposed, where the mucus matrix containing acidic binding sites acts as an intercellular glue, providing a molecular sieve-like filtering capacity to the aggregates dispersed in seawater.