Alpha 2HS Research Articles

Fetuin-A Promotes 3-Dimensional Growth in LNCaP Prostate Cancer Cells by Sequestering Extracellular Vesicles to Their Surfaces to Act as Signaling Platforms.

The present studies were conducted to evaluate key serum proteins and other components that mediate anchorage-independent growth (3-D growth) of LNCaP prostate cancer cells as spheroids. The cells were cultured on ultra-low attachment plates in the absence and presence of fetuin-A and with or without extracellular vesicles. The data show that fetuin-A (alpha 2HS glycoprotein) is the serum protein that mediates 3-D growth in these cells. It does so by sequestering extracellular vesicles of various sizes on the surfaces of rounded cells that grow as spheroids. These vesicles in turn transmit growth signals such as the activation of AKT and MAP kinases in a pattern that differs from the activation of these key growth signaling pathways in adherent and spread cells growing in 2-D. In the process of orchestrating the movement and disposition of extracellular vesicles on these cells, fetuin-A is readily internalized in adhered and spread cells but remains on the surfaces of non-adherent cells. Taken together, our studies suggest the presence of distinct signaling domains or scaffolding platforms on the surfaces of prostate tumor cells growing in 3-D compared to 2-D.

Bioinformatic analysis of proteomic data for iron, inflammation, and hypoxic pathways in restless legs syndrome

Objective/BackgroundWe performed bioinformatic analysis of proteomic data to identify the biomarkers of restless legs syndrome (RLS) and provide insights into the putative pathomechanisms, including iron deficiency, inflammation, and hypoxic pathways. Patients/methodsPatients with drug-naïve idiopathic RLS were recruited at a university hospital from June 2017 to February 2018. Serum samples from patients with RLS (n = 7) and healthy sex- and age-matched controls (n = 6) were evaluated by proteomic analysis. For differentially expressed proteins (DEPs) in patients with RLS, compared to those in controls, the expression profiles and protein–protein interaction (PPI) network were characterized between dysregulated proteins and extracted proteins involved in iron deficiency, hypoxia, and inflammation responses using the String database (http://string-DB.org). The PPI network was visualized by Cytoscape ver. 3. 7. 1. Statistical analyses of the validation Western blot assays were performed using a Student's t-test. ResultsInteractome network analysis revealed a relationship among the eight proteins, their associated genes, and 150, 47, and 11 proteins related to iron deficiency, inflammation, and hypoxic pathways, respectively. All DEPs were well associated with inflammation, and complement 3, complement C4A, alpha-2 HS glycoprotein, and alpha-2 macroglobulin precursor were found to be in hub positions of networks involved in PPIs including iron deficiency, hypoxia pathway, and inflammation. C3 and C4A were verified using western blotting. ConclusionsWe identified key molecules that represent the selected cellular pathways as protein biomarkers by PPI network analysis. Changes in inflammation can mediate or affect the pathomechanism of RLS and can thus act as systemic biomarkers.

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.

The interactions of alpha 2HS glycoprotein with metalloproteinases.

The objective of the present analyses was to determine whether matrix metalloproteinases interact with alpha 2HS glycoprotein, the human homologue of bovine fetuin. Alpha 2HS glycoprotein was incubated with metalloproteinases at 1:1 and 1:10 molar ratios. The serum glycoprotein was completely degraded by gelatinase-A and matrilysin at the higher enzyme concentration after an overnight incubation at 37 degrees C. The data show that gelatinase-A, matrilysin and gelatinase-B do interact with alpha 2HS glycoprotein and that gelatinase-B associates most tightly with the serum glycoprotein.

Calcium-binding properties of fetuin in fetal bovine serum.

Fetuin, an abundant protein in fetal bovine plasma, is the bovine homolog of human alpha 2HS glycoprotein (alpha 2HS). In spite of numerous studies, the biological functions of both proteins remain elusive. We now report the remarkable 45Ca-binding activity of fetuin in fetal bovine serum that has been blotted on a membrane after electrophoresis. The Ca-binding ability of the purified protein was analyzed by equilibrium dialysis, which revealed that bovine fetuin had multiple Ca-binding sites, one of which had a Kd of 0.95 x 10(-4) M. It was also shown that the Ca-binding activity of fetuin was greater than that of albumin in serum of the bovine fetus at the late gestational stage. Since fetal bovine serum contains not only a high concentration of fetuin but also a high concentration of Ca, it is possible that fetuin functions to maintain high levels of Ca in fetal serum via normalization of the concentration of Ca2+ ions.

Distribution of α2Hs Glycoprotein in Pathological Human Dentins

The concentration and distribution of alpha 2HS glycoprotein in teeth from patients with heritable dentin disorders were examined utilizing immunological techniques. ELISA for the quantitative determination showed lower concentrations of alpha 2HS glycoprotein in dentins of patients with heritable disorders than in the control; some cases contained very low and others intermediate concentrations. Immunohistochemical findings indicated that the concentration of this protein was correlated to the regularity of the dentinal tubules. This result suggests that alpha 2HS glycoprotein is incorporated into dentin in concomitance with the development of the dentinal tubule, especially with the deposition of peritubular dentin. Incorporation of this protein into dentin is thought to involve the function of odontoblasts.

Fetuin--an old friend revisited.

Bovine fetuin, the first fetal protein to be described, has recently been shown to be a species homologue of a well known human plasma protein--alpha 2HS glycoprotein (alpha 2HS). The fetuins are now known to be members of the cystatin superfamily. The structural properties of the six fetuins that have been fully sequenced are compared. Despite the structural homology of these proteins, their described properties in the literature make them appear to be quite different. The diverse in vitro properties claimed for fetuin/alpha 2HS are reviewed. In vivo, fetuins are involved in the acute phase response. In development, in all species studied so far, fetuins are present in a specific cell population that forms the developing neocortex. The possible functional significance of this distribution is discussed.

Fetuin and alpha‐2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.

Genetic polymorphism of Alpha-2-HS glycoprotein in Lombardy (Italy)

Alpha 2HS-subtypes have been analysed in samples from 700 unrelated individuals from the Brescia area (Italy) by the isoelectric focusing technique and immunofixation. The observed allele frequencies were: alpha 2HS*1 = 0.7472; alpha 2HS*2 = 0.2507; alpha 2HS*V = 0.0021.

Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenii)

A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of FLG in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and alpha 2HS glycoprotein in human fetal brain. The sequence of appearance of FLG-positive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of FLG in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed.

Bone matrix constituents stimulate interleukin-1 release from human blood mononuclear cells.

To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis.

Tooth ‘enamelins’ identified mainly as serum proteins

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with polyclonal and monoclonal antibodies to albumin. Two-dimensional separation of the components in the enamelin extract by isoelectric focusing and SDS/PAGE reveal that the major approximately 67-kDa component and almost all of the minor Coomassie-staining protein components of approximately 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The approximately 67-kDa band eluted after SDS/PAGE, as well as the major approximately 67-kDa spots eluted after two-dimensional separation, were found to have N-terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70-80% of the total protein content of the enamelin extract and was essentially the only protein in the approximately 67-kDa component. The serum proteins alpha-2 HS glycoprotein, gamma-globulin and fetuin, and the proline-rich salivary protein termed P-B were also identified in the enamelin extract. The serum proteins and the salivary protein account for greater than 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non-serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N-terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true 'enamelins' synthesized by ameloblasts. One additional protein had the first five N-terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N-terminally blocked on attempted sequencing. None of the components in the neutral soluble low-ionic-strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti-albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain non-amelogenin proteins. On the basis of the data presented, studies employing antibodies to the so-called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed.

Immunohistochemical localization of α2HS glycoprotein in dentinHS glycoprotein in dentin

Monoclonal antibody against bovine serum alpha 2HS glycoprotein was raised in mice and the localization of this glycoprotein in bovine and human teeth was examined by immunohistochemical techniques on undemineralized ground sections after surface etching with dilute HCl. Positive reactions were found in both bovine and human peritubular dentin, whereas virtually negative staining was detected in the intertubular dentin. When the ground section was examined without surface etching, no reaction was found in any area of mineralized dentin. These results suggest that alpha 2HS glycoprotein is present mainly in the peritubular dentin associated with the mineral phase. The presence of this component in dentin may not be due simply to nonspecific binding to calcium phosphate crystals.

The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily.

The cDNA sequence encoding the bovine fetal protein fetuin is reported. The deduced amino acid sequence is identical with that obtained from amino acid sequencing. The protein is a single chain preceded by a signal sequence. The three N-linked glycosylation sites have been determined. The sequence of fetuin shows over 70% similarity to human alpha 2HS glycoprotein. All of the cysteine residues are conserved in both proteins, suggesting that fetuin has the same arrangement of disulfide loops as alpha 2HS glycoprotein and may also be a member of the cystatin family. Southern blot analysis indicates that a single gene codes for fetuin. No evidence for a separate gene for a bovine alpha 2HS glycoprotein was obtained; thus, fetuin in cattle and alpha 2HS glycoprotein in the human are equivalent proteins.

Serum Gc, Hp and &amp;alpha;&lt;sub&gt;2&lt;/sub&gt;HS Phenotypes in Human T-Lymphotropic Leukemia Virus Type I Infection

The serum Gc, Hp and alpha 2HS phenotypes were examined in 64 subjects known to have the human T-lymphotropic leukemia virus type I (HTLV-I) infection and in 60 uninfected subjects. There were no significant differences in the distributions of Gc, Hp and alpha 2HS phenotype and allele frequencies between any grouping of HTLV-I-infected subjects and the controls. No association between the Gc, Hp and alpha 2HS genotypes and susceptibility to adult T-cell leukemia was found.

Changes in various serum protein levels between acute and convalescent phases in patients with respiratory infections

We determined serum levels of alpha 1-antitrypsin, alpha 1-acid glycoprotein, ceruloplasmin, haptoglobin, alpha 2-HS glycoprotein, C3 and fibronectin in 22 patients with acute pneumonia, 10 patients with mycoplasma pneumonia and 8 patients with exacerbation of chronic bronchitis at both acute and convalescent phases to clarify whether levels of these proteins change with inflammation. Serum levels of alpha 1-antitrypsin, alpha 1-acid glycoprotein, ceruloplasmin, haptoglobin and C3 in the patients in the acute phase were significantly higher than that in the patients in the convalescent phase. The serum level of fibronectin in the patients in the acute phase was significantly lower than that in the patients in convalescent phase. The serum level of alpha 2HS glycoprotein remained unchanged.

Separation of bone matrix proteins by calcium-induced precipitation

It was found that significant precipitation occurred immediately after calcium, at a concentration as low as 2 mM, was added to a desalted solution of EDTA extract of adult bovine femur. The maximal yield of the precipitates was observed at a calcium concentration of 30 mM. These precipitates were dissolved in 0.5 M EDTA, desalted, and characterized by Sepharose CL-6B gel filtration chromatography and high performance gel-exclusion chromatography. Results revealed that the precipitates were enriched in a 40 K protein and a higher molecular weight fraction as compared with the original extract of bone proteins. The 40 K fraction was isolated and identified as osteonectin, as judged from amino acid analysis, electrophoresis, and immunodetection. The supernatant after calcium-induced precipitation predominantly contained osteocalcin and a 50 K protein that was tentatively identified as alpha 2HS protein. Osteonectin was purified from the calcium-induced precipitates from the EDTA extract of bovine bone. By calcium titration using fluorescence spectrometry, the isolated osteonectin showed high affinity to calcium ions with an apparent dissociation constant (K0.5) of 8 x 10(-7) M. Thus, the use of calcium to separate bone proteins, especially osteonectin, was proved to be a useful technique. In addition, calcium-induced precipitation of osteonectin suggested a possible in vivo mechanism via which osteonectin might interact with calcium ions and participate in the initial immobilization of calcium to induce the nucleation of calcification in bone tissue.

Major “Enamelin” Protein in Enamel of Developing Bovine Teeth is Albumin

The major, non-amelogenin protein component (enamelin) present in EDTA or EDTA-GU HCl extracts of developing bovine enamel has a molecular weight of approximately 67 kD and an amino acid composition rich in asp, glu, ala, leuc and lys. Elution of this component from 1.5 and 3.0 mm thick strips SDS-PAGE gels and subsequent analyses show that the component selectively reacts with polyclonal antibody to bovine serum albumin. Absolute identification of this major enamelin component as serum albumin is established by an amino acid sequence of the first 40 N-Terminal amino acids which was found to be identical to bovine serum albumin. In addition to albumin, alpha-2 HS glycoprotein was also identified in the same extracts by Western blotting against a monospecific polyclonal antibody against human alpha-2 HS glycoprotein.

Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.

Three forms of BRP-2 (bone resorptive proteins) from human cancer ascites fluid and their relationship to human serum alpha-2 HS-glycoprotein

Two new forms of BRP-2, a previously described bone resorptive protein, were purified from ascites fluids obtained from patients with hypercalcemia and metastatic bone cancer. The apparent molecular weights of BRP-2 and of these two proteins were 52,000, 48,000, and 46,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The three proteins have essentially the same amino acid compositions but differ with respect to their carbohydrate moieties. The amino-terminal amino acid sequences of the three glycoproteins were identical to each other as well as to human serum alpha 2HS-(human serum) glycoprotein. The relationship of the three forms of BRP-2 to alpha 2HS was also established immunochemically. The ascites proteins, as well as alpha 2HS, on a molar basis, were approximately one-tenth as potent as bovine parathyroid hormone fragment (1-34) in their abilities to stimulate calcium release from bone in vitro. This study describes for the first time a possible function for human serum alpha 2HS.