Thermodynamic Evolution of a Metamorphic Protein: A Theoretical-Computational Study of Human Lymphotactin.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

Derivatization of sialylated glycopeptides plus based sialoglycopeptides enrichment using cation exchange media.

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Assessing the Performance of Traveling-salesman based Automated Path Searching (TAPS) on Complex Biomolecular Systems.

Though crucial for understanding the function of large biomolecular systems, locating the minimum free energy paths (MFEPs) between their key conformational states is far from trivial due to their high-dimensional nature. Most existing path-searching methods require a static collective variable space as input, encoding intuition or prior knowledge of the transition mechanism. Such information is, however, hardly available a priori and expensive to validate. To alleviate this issue, we have previously introduced a Traveling-salesman based Automated Path Searching method (TAPS) and demonstrated its efficiency on simple peptide systems. Having implemented a parallel version of this method, here we assess the performance of TAPS on three realistic systems (tens to hundreds of residues) in explicit solvents. We show that TAPS successfully located the MFEP for the ground/excited state transition of the T4 lysozyme L99A variant, consistent with previous findings. TAPS also helped identifying the important role of the two polar contacts in directing the loop-in/loop-out transition of the mitogen-activated protein kinase kinase (MEK1), which explained previous mutant experiments. Remarkably, at a minimal cost of 126 ns sampling, TAPS revealed that the Ltn40/Ltn10 transition of lymphotactin needs no complete unfolding/refolding of its ß-sheets and that five polar contacts are sufficient to stabilize the various partially unfolded intermediates along the MFEP. These results present TAPS as a general and promising tool for studying the functional dynamics of complex biomolecular systems.

Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis.

Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.

Structural basis for transcriptional coactivator recognition by SMAD2 in TGF-ß signaling.

Transforming growth factor-ß (TGF-ß) proteins regulate multiple cellular functions, including cell proliferation, apoptosis, and extracellular matrix formation. The dysregulation of TGF-ß signaling causes diseases such as cancer and fibrosis, and therefore, understanding the biochemical basis of TGF-ß signal transduction is important for elucidating pathogenic mechanisms in these diseases. SMAD proteins are transcription factors that mediate TGF-ß signaling-dependent gene expression. The transcriptional coactivator CBP directly interacts with the MH2 domains of SMAD2 to activate SMAD complex-dependent gene expression. Here, we report the structural basis for CBP recognition by SMAD2. The crystal structures of the SMAD2 MH2 domain in complex with the SMAD2-binding region of CBP showed that CBP forms an amphiphilic helix on the hydrophobic surface of SMAD2. The expression of a mutated CBP peptide that showed increased SMAD2 binding repressed SMAD2-dependent gene expression in response to TGF-ß signaling in cultured cells. Disrupting the interaction between SMAD2 and CBP may therefore be a promising strategy for suppressing SMAD-dependent gene expression.

USPIO-PEG nanoparticles functionalized with a highly specific collagen-binding peptide: a step towards MRI diagnosis of fibrosis.

Fibrosis is characterized by a pathologic deposition of collagen I, leading to impaired function of organs. Tissue biopsy is the gold standard method for the diagnosis of fibrosis but this is an invasive procedure, subject to sampling errors. Several non-invasive techniques such as magnetic resonance imaging (MRI) using non-specific probes have been developed but they are not fully satisfying as they allow diagnosis at a late stage. In this study, collagelin, a collagen-binding peptide has been covalently linked using click chemistry to pegylated Ultra Small Super Paramagnetic Iron Oxide Nanoparticles (USPIO-PO-PEG-collagelin NPs) with the aim of diagnosing fibrosis at an early stage by MRI. USPIO-PO-PEG-collagelin NPs showed a high affinity for collagen I, two times higher than that of free collagelin whereas not peptide labeled USPIO NPs (USPIO-PO-PEG-yne) did not present any affinity. NPs were not toxic for macrophages and fibroblasts. Diffusion through collagen hydrogels concentrated at 3 and 10 mg mL-1 revealed a large accumulation of USPIO-PO-PEG-collagelin NPs within the collagen network after 72 hours, ca. 3 times larger than that of unlabeled USPIO, thereby evidencing the specific targeting of collagen I. Moreover, the quantity of USPIO-PO-PEG-collagelin NPs accumulated within hydrogels was proportional to the collagen concentration. Subsequently, the NPs diffusion through collagen hydrogels was monitored by MRI. The MRI T2 time relaxation decreased much more significantly with depth for USPIO-PO-PEG-collagelin NPs compared to unlabeled ones. Taken together, these results show that USPIO-PEG-collagelin NPs are promising as effective MRI nanotracers for molecular imaging of fibrosis at an early stage.

Immunohistochemical study of dental pulp cells with 3D collagen type I gel in demineralized dentin tubules in vivo.

Dental pulp cells (DPCs) represent good candidates for the regeneration of dental tissue. This study aimed to evaluate the growth and differentiation potential of DPCs cultured inside demineralized dentin tubules in vivo. Six green fluorescent protein-transgenic rats (body weight 100 g each) and thirty-two Sprague-Dawley (SD) male rats (body weight 250 g each) were used for DPC collection and dentin tubules preparation and transplantation, respectively. Third-passage DPCs with or without collagen gels were loaded into demineralized dentin tubules. Both types of grafts were transplanted into the rectus abdominis muscles of SD rats and were harvested after 21 days. The expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (OPN), nestin, and dentin sialoprotein (DSP) was analyzed by immunohistochemistry. Histological analysis showed that DPCs in the collagen gel formed an osteodentin-like hard tissue matrix after 21 days. Increased positive immunoreactivity for ALP, BSP, OPN, nestin, and DSP was observed in experimental groups compared with control. Our results demonstrate that DPCs in collagen gel inside demineralized dentin tubules show increased growth and differentiation.

Natural protein bioinspired materials for regeneration of hard tissues.

The regenerative materials for hard tissues, i.e. tooth (enamel, dentin, and cementum) and bone, require extremely high standards in terms of their mechanical properties, biocompatibility, bioactivity, and multiple-functionality. Among them, the biomedical materials inspired from various natural proteins have attracted increasing research attention. These blueprint proteins include various hard-tissue-related proteins, such as collagen and non-collagenous proteins (e.g. amelogenin, dentin phosphoprotein, bone sialoprotein, and osteopontin), as well as other natural proteins like mussel foot proteins. The current review highlights the structure-function relationship of protein bioinspired biomedical materials (e.g. polymers and polypeptides) and their applications for tooth and bone regeneration. Specifically, the materials bioinspired from salivary acquired pellicle proteins, which have a strong affinity to hydroxyapatite surfaces, are discussed in detail. Finally, the challenges associated with these protein bioinspired materials and their industrialization potentials are discussed.

CBP mediated DOT1L acetylation confers DOT1L stability and promotes cancer metastasis.

Background and Aim: DOT1L regulates various genes involved in cancer onset and progression by catalyzing H3K79 methylation, but how DOT1L activity itself is regulated is unclear. Here, we aimed to identify specific DOT1L post-translational modifications that might regulate DOT1L activity and thus impact on colorectal cancer (CRC) progression. Methods: We conducted affinity purification and mass spectrometry to explore DOT1L post-translational modifications. We then established transwell migration and invasion assays to specifically investigate the role of DOT1L(K358) acetylation on CRC cellular behavior in vitro and a bioluminescence imaging approach to determine the role of DOT1L(K358) acetylation in CRC metastasis in vivo. We performed chromatin immunoprecipitation to identify DOT1L acetylation-controlled target genes. Finally, we used immunohistochemical staining of human tissue arrays to examine the relevance of DOT1L(K358) acetylation in CRC progression and metastasis and the correlation between DOT1L acetylation and CBP. Results: We found that CBP mediates DOT1L K358 acetylation in human colon cancer cells and positively correlates with CRC stages. Mechanistically, DOT1L acetylation confers DOT1L stability by preventing the binding of RNF8 to DOT1L and subsequent proteasomal degradation, but does not affect its enzyme activity. Once stabilized, DOT1L can catalyze the H3K79 methylation of genes involved in epithelial-mesenchymal transition, including SNAIL and ZEB1. An acetylation mimic DOT1L mutant (Q358) could induce a cancer-like phenotype in vitro, characterized by metastasis and invasion. Finally, DOT1L(K358) acetylation correlated with CRC progression and a poor survival rate as well as with high CBP expression. Conclusions: DOT1L acetylation by CBP drives CRC progression and metastasis. Targeting DOT1L deacetylation signaling is a potential therapeutic strategy for DOT1L-driven cancers.

9-Azido Analogues of Three Sialic Acid Forms for Metabolic Remodeling of Cell-Surface Sialoglycans.

Neu5Ac, Neu5Gc, and KDN are three forms of sialic acids in vertebrates that possess distinct biological functions. Herein, we report the synthesis and metabolic incorporation of the 9-azido analogues of three sialic acid forms in mammalian cells. The incorporated sialic acid analogues enable fluorescent imaging of cell-surface sialoglycans and proteomic profiling of sialoglycoproteins. Furthermore, we apply them to metabolically engineer cell surfaces with sialoglycans terminated with distinct sialic acids or their 9-azido analogues. The remodeled cells expressing specific cell-surface sialoglycoforms show distinct binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted by Shiga toxigenic Escherichia coli. The 9-azido analogues of sialic acid forms developed in this work provide a versatile tool for metabolic remodeling of cell-surface properties and modulating pathogen-host interactions.

Identification of Cryptosporidium viatorum XVa subtype family in two wild rat species in China.

BACKGROUND: Cryptosporidium viatorum is a minor Cryptosporidium pathogen in humans. Currently, there is limited information regarding the prevalence and genotypes of C. viatorum in animals in China. METHODS: In this study, 228 faecal samples were collected from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in Chongqing Municipality and Guangdong Province, China. These specimens were analyzed for C. viatorum and then subtyped it using PCR and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) and 60-kilodalton glycoprotein (gp60) genes, respectively. RESULTS: A total of 25 (11.0%) faecal samples were tested positive for C. viatorum by SSU rRNA assay. Of these samples, 4 (3.6%) came from L. edwardsi and 21 (18.0%) from B. bowersi. Of the 25 C. viatorum-positive samples, 17 were successfully amplified at the gp60 gene locus, which represented four subtypes belonging to two subtype families, including XVa (XVaA6, XVaA3g, XVaA3h) and XVc (XVcA2G1). Phylogenetic analysis based on the gp60 amino acid sequences indicated that all of the C. viatorum isolates grouped together, supporting the conclusion that C. viatorum from the wild rats represent two subtype families. CONCLUSIONS: These results indicate an occurrence of C. viatorum XVa subtype family from rats which is genetically identical to those found in humans. Our findings suggest that wild rats may be a potential source of human cryptosporidiosis.

Preparation and Characterization of Dentin Phosphophoryn-Derived Peptide-Functionalized Lignin Nanoparticles for Enhanced Cellular Uptake.

The surface modification of nanoparticles (NPs) using different ligands is a common strategy to increase NP-cell interactions. Here, dentin phosphophoryn-derived peptide (DSS) lignin nanoparticles (LNPs) are prepared and characterized, the cellular internalization of the DSS-functionalized LNPs (LNPs-DSS) into three different cancer cell lines is evaluated, and their efficacy with the widely used iRGD peptide is compared. It is shown that controlled extent of carboxylation of lignin improves the stability at physiological conditions of LNPs formed upon solvent exchange. Functionalization with DSS and iRGD peptides maintains the spherical morphology and moderate polydispersity of LNPs. The LNPs exhibit good cytocompatibility when cultured with PC3-MM2, MDA-MB-231, and A549 in the conventional 2D model and in the 3D cell spheroid morphology. Importantly, the 3D cell models reveal augmented internalization of peptide-functionalized LNPs and improve antiproliferative effects when the LNPs are loaded with a cytotoxic compound. Overall, LNPs-DSS show equal or even superior cellular internalization than the LNPs-iRGD, suggesting that DSS can also be used to enhance the cellular uptake of NPs into different types of cells, and release different cargos intracellularly.

Identification of Multisialylated LacdiNAc Structures as Highly Prostate Cancer Specific Glycan Signatures on PSA.

Serum prostate-specific antigen (PSA) test is the current gold standard for screening and diagnosis of prostate cancer (PCa), while overdiagnosis and overtreatment are social problems. In order to improve the specificity and exclude a false positive diagnosis in PSA test, PCa-specific glycosylation subtypes of PSA were explored using in-depth quantitative profiling of PSA glycoforms based on mass spectrometric oxonium ion monitoring technology. As a result of analysis using sera from 15 PCa or 15 benign prostate hyperplasia (BPH) patients whose PSA levels were in the "gray zone" (4.0-10.0 ng/mL), 52 glycan structures on PSA were quantitatively observed. We found that abundance of multisialylated LacdiNAc (GalNAcß1-4GlcNAc) structures were significantly upregulated in the PCa group compared to the BPH group. A couple of those glycoforms were then extracted and subjected to establish a novel PCa-specific diagnosis model (PSA G-index). When the diagnostic power was assessed using an independent validation sample set (15 PCa and 15 BPH patients in the PSA gray zone), an AUC of PSA G-index was 1.00, while that of total PSA or PSA f/T ratio was 0.50 or 0.60, respectively. Moreover, both PSA glycoforms showed significant correlation with Gleason scores. Lectin histochemical staining analysis also showed that PCa cells overexpressed glycoproteins containing LacdiNAc and sialic acids moieties. Thus, PSA G-index could serve as not only an effective secondary screening method to exclude false positive diagnosis in PSA screening, but also a potential grading biomarker for PCa.

Dentin Phosphoprotein Mimetic Peptide Nanofibers Promote Biomineralization.

Dentin phosphoprotein (DPP) is a major component of the dentin matrix playing crucial role in hydroxyapatite deposition during bone mineralization, making it a prime candidate for the design of novel materials for bone and tooth regeneration. The bioactivity of DPP-derived proteins is controlled by the phosphorylation and dephosphorylation of the serine residues. Here an enzyme-responsive peptide nanofiber system inducing biomineralization is demonstrated. It closely emulates the structural and functional properties of DPP and facilitates apatite-like mineral deposition. The DPP-mimetic peptide molecules self-assemble through dephosphorylation by alkaline phosphatase (ALP), an enzyme participating in tooth and bone matrix mineralization. Nanofiber network formation is also induced through addition of calcium ions. The gelation process following nanofiber formation produces a mineralized extracellular matrix like material, where scaffold properties and phosphate groups promote mineralization. It is demonstrated that the DPP-mimetic peptide nanofiber networks can be used for apatite-like mineral deposition for bone regeneration.

Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs.

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. ß-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Fast screening of N-glycosylation disorders by sialotransferrin profiling with capillary zone electrophoresis.

Background Congenital disorders of glycosylation (CDG) are a growing group of rare genetic disorders. The most frequently used screening method is sialotransferrin profiling using isoelectric focusing (IEF). Capillary zone electrophoresis (CZE) may be a simple and fast alternative. We investigated the Capillarys™ CDT assay (Sebia, France) to screen for N-glycosylation disorders, using IEF as gold standard. Methods Intra- and inter-assay precision were established, and analyses in heparin-anticoagulated plasma and serum were compared. Accuracy was assessed by comparing IEF and CZE profiles of 153 samples, including 49 normal, 53 CDG type I, 2 CDG type II, 1 combined CDG type I and type II and 48 samples with a Tf-polymorphism. Neuraminidase-treated plasma was analysed to discriminate CDG and Tf-polymorphisms using samples of 52 subjects (25 had a confirmed Tf-polymorphism). Age-dependent reference values were established using profiles of 312 samples. Results Heparin-plasma is as suitable as serum for CDG screening with the Capillarys™ CDT assay. The precision of the method is high, with a limit of quantification (LOQ) of 0.5%. All profiles, including CDG and Tf-polymorphisms, were correctly identified with CZE. Forty-nine of 52 neuraminidase-treated samples correctly identified the presence/absence of a Tf-polymorphism. Interferences in 3/52 samples hampered interpretation. Sialo-Tf profiles were dependent of age, in particular in the first three months of age. Conclusions CZE analysis with the Capillarys™ CDT kit (Sebia) is a fast and reliable method for screening of N-glycosylation defects. Tf-polymorphisms could be excluded after overnight incubation with neuraminidase.

Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using IntactGIG-HILIC.

Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. Graphical Abstract ᅟ.

Molecular mechanism of K65 acetylation-induced attenuation of Ubc9 and the NDSM interaction.

The negatively charged amino acid-dependent sumoylation motif (NDSM) carries an additional stretch of acidic residues downstream of the consensus Ψ-K-x-E/D sumoylation motif. We have previously shown that acetylation of the SUMO E2 conjugase enzyme, Ubc9, at K65 downregulates its binding to the NDSM and renders a selective decrease in sumoylation of substrates with the NDSM motif. Here, we provide detailed structural, thermodynamic, and kinetics results of the interactions between Ubc9 and its K65 acetylated variant (Ac-Ubc9K65) with three NDSMs derived from Elk1, CBP, and Calpain2 to rationalize the mechanism beneath this reduced binding. Our nuclear magnetic resonance (NMR) data rule out a direct interaction between the NDSM and the K65 residue of Ubc9. Similarly, we found that NDSM binding was entropy-driven and unlikely to be affected by the negative charge by K65 acetylation. Moreover our NMR, mutagenesis and molecular dynamics simulation studies defined the sequence of the NDSM as Ψ-K-x-E/D-x1-x2-(x3/E/D)-(x4/E/D)-xn and determined that K74 and K76 were critical Ubc9 residues interacting with the negatively charged residues of the NDSM.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement.

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.