Akraporn Prakobphol

Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface

Trophoblasts, the specialized cells of the placenta, play a major role in implantation and formation of the maternal-fetal interface. Through an unusual differentiation process examined in this review, these fetal cells acquire properties of leukocytes and endothelial cells that enable many of their specialized functions. In recent years a great deal has been learned about the regulatory mechanisms, from transcriptional networks to oxygen tension, which control trophoblast differentiation. The challenge is to turn this information into clinically useful tests for monitoring placental function and, hence, pregnancy outcome.

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Sweetening the Pot: Adding Glycosylation to the Biomarker Discovery Equation

BACKGROUND Cancer has profound effects on gene expression, including a cells glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies--multiple reaction monitoring and lectin-antibody arrays--as potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.

Binding of the Streptococcal Surface Glycoproteins GspB and Hsa to Human Salivary Proteins

ABSTRACT GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibα. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.

MUC1 is a scaffold for selectin ligands in the human uterus.

MUC1 is a large, transmembrane mucin glycoprotein abundantly expressed at the apical surface of uterine epithelia in all species examined to date. Loss of MUC1 at the time of embryo implantation occurs in many species; however, this does not appear to be the case in humans. Recent studies indicate that human blastocysts express L-selectin at their external surfaces raising the possibility that selectin ligands expressed at the apical surface of the uterine epithelium support early stages of blastocyst attachment. In the current study, we have used a panel of antibodies specific for selectin ligands to determine if MUC1 functions as a scaffold for these carbohydrate motifs in fertile women. The results demonstrate that MUC1 carries selectin ligands throughout the secretory phase of the menstrual cycle, including the mid-secretory (receptive) phase. Consequently, MUC1 represents a potential ligand for selectins expressed by human blastocysts.

The High-Molecular-Weight Human Mucin Is the Primary Salivary Carrier of ABH, Lea, and Leb Blood Group Antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples.

Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope‐based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C‐terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof‐of‐principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.

Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis.

Human trophoblast progenitors: where do they reside?

In humans, very little is known about the factors that regulate trophoblast (TB) specification, expansion of the initial TB population, and formation of the cytotrophoblast (CTB) populations that populate the chorionic villi. The absence of human trophoblast progenitor cell (hTPC) lines that can be propagated in vitro has been a limiting factor. Because attempts to derive TB stem cells from the trophectoderm of the human blastocyst have so far failed, investigators use alternative systems as cell culture models including TBs derived from human embryonic stem cells (hESCs), immortalized CTBs, and cell lines established from TB tumors. Additionally, the characteristics of mature TBs have been extensively studied using primary cultures of CTBs and explants of placental chorionic villi. However, none of these models can be used to study TB progenitor self-renewal and differentiation. Furthermore, the propagation of human TB progenitors from villous CTBs (vCTBs) has not been achieved. The downregulation of key markers of cell cycle progression in vCTBs by the end of the first trimester of pregnancy may indicate that these cells are not a source of human TB progenitors later in pregnancy. In contrast, mesenchymal cells of the villi and chorion continue to proliferate until the end of pregnancy. We recently reported isolation of continuously self-renewing hTPCs from chorionic mesenchyme and showed that they differentiated into the mature TB cell types of the villi, evidence that they can function as TB progenitors. This new cell culture model enables a molecular analysis of the seminal steps in human TB differentiation that have yet to be studied in humans. In turn, this information can be used to trace the origins of pregnancy complications that are associated with faulty TB growth and differentiation.