Extraction of heavy metals from wastewater using amine-modified mesoporous silica.

Presence of heavy metals in wastewater is a critical environmental issue, and efficient extraction of the metals remains a challenging task. In this study, the adsorption behavior of Ce(III), Hg(II), and Cu(II) metal ions using MCM-48 material modified with acid and base functional groups was examined. The modified materials were characterized using various techniques, including XRD, BET, FT-IR, NMR, and SEM, which revealed that the materials' properties remained unchanged after modification. The adsorption capacity of the modified materials for metal ions was then evaluated and was found that the amine-modified MCM-48 material exhibited the highest adsorption efficiency. Precisely, the amine-modified material achieved an adsorption capacity of 97% for Ce(III), 98% for Hg(II), and 90% for Cu(II) after 180 min of adsorption. These results highlight the effectiveness of amine functionalization in enhancing the adsorption capacity of silica material for heavy metals.

Ni4 complex anchored porous silica for enhanced adsorption of organic pollutants in the wastewater.

In the current study, tetranuclear Ni complex [Ni4(LH)4]·CH3CN (1) (LH3=(E)-2-(hydroxymethyl)-6-(((2-hydroxyphenyl)imino)methyl)phenol) was prepared and incorporated in sulfonic acid functionalized MCM-48 material. This composite nanoporous material was investigated for the adsorption of toxic cationic water pollutant dyes like crystal violet (CV) and methylene blue (MB) from the water solution. Thorough characterization was carried out using a variety of techniques, including NMR, ICP, powder XRD, TGA, SEM, BET, and FT-IR, to verify the phase purity, existence of guest moiety, material morphology, and other crucial variables. The adsorption property was increased with the metal complex immobilization on the porous support. The effect of various parameters on the adsorption process was discussed, including adsorbent dosage, temperature, pH, NaCl concentration, and contact time. Maximum dye adsorption was found at 0.2 mg/ml adsorbent dosage, 10 ppm dye concentration, 6-7 pH, 25 °C temperature, and 15 minutes of contact time. The adsorption of MB (methylene blue) and CV (crystal violet) dyes by Ni complex integrated MCM-48 was effective, with over 99% adsorption achieved in 15 minutes. A recyclability test was also performed, and the material is reusable up to the third cycle, with no notable decline in adsorption found. From the previous literature survey, it is clear that very high adsorption efficiency was achieved using MCM-48-SO3-Ni in considerably short contact time which proves the novelty and effectiveness of the modified material. Ni4 was prepared, characterized, and immobilized in sulfonic acid functionalized MCM-48, and this robust and reusable adsorbent was highly effective for the adsorption of methylene blue and crystal violet dyes with >99% adsorption efficiency in short duration.

Data on ultrasound therapy as an adjuvant pain control method among Indian TMDS patients.

It is of interest to evaluate the efficacy of ultrasound therapy as an adjuvant pain control modality in dysfunctions of the temporomandibular joint. The study comprised 20 patients with TMJ issues who had received a clinical diagnosis of temporomandibular disorders (TMDS). These patients underwent independent VAS evaluations for the intensity of pain, opening and closing of the mouth, and soreness of the muscles of mastication, including the masseter muscle, medial pterygoid muscle, lateral pterygoid muscle, and temporalis muscle, as well as additional auxiliary muscles. The chosen patients received ultrasonic treatment. The mean value of mouth opening before therapy was 39.51cm, with SD values of 7.61 cm. The mean value of mouth opening after therapy was 42.91 cm with SD values of 6.08.The findings were statistically significant, with a p-valueof0.021. The mean value of VAS in the TMJ area before therapy was 8.41 with SD values of 2.11.There was a reduction in the mean values of VAS after therapy, which was 3.11 with SD values of 1.12. The findings were significant statistically, with a p-value equal to 0.001. Thus, ultrasonographic therapy for temporomandibular joint pain demonstrated a considerable improvement in pain reduction and mouth opening. It is possible to view this therapy as the adjuvant methodology to control pain in disorders of TMJ.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells.

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss.

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.

km23-1/DYNLRB1 regulation of MEK/ERK signaling and R-Ras in invasive human colorectal cancer cells.

We previously found that km23-1/DYNLRB1 is required for transforming growth factor-ß (TGFß) production through Ras/ERK pathways in TGFß-sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23-1/DYNLRB1 is required for mitogen-activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23-1/DYNLRB1-siRNA inhibition of phospho-(p)-MEK immunostaining in RKO cells. Furthermore, we show that CRISPR-Cas9 knock-out (KO) of km23-1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD-1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFß-mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFß-mediated activation of MEK1/2 or c-Jun N-terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B-Raf, extracellular signal-regulated kinase (ERK), and p-ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23-1/DYNLRB1 co-sedimented with Ras, p-ERK, and ERK in fractions that did not contain components of holo-dynein. Thus, km23-1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein-independent km23-1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R-Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23-1/DYNLRB1 and RRas wase co-localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23-1/DYNLRB1-R-Ras complex in CRC invasion.

Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs.

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068-79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.

HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs.

The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.

A hyaluronan receptor for endocytosis (HARE) link domain N-glycan is required for extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) signaling in response to the uptake of hyaluronan but not heparin, dermatan sulfate, or acetylated low density lipoprotein (LDL).

The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn(2280)), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991-1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756-1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.

Evaluation of awareness regarding orthodontic procedures among a group of preadolescents in a cross-sectional study.

OBJECTIVE: This study was carried out to know the level of awareness regarding orthodontic procedures among preadolescents as there is very high prevalence of malocclusion. METHODS: This cross-sectional study was conducted among a sample of 1010 subjects with a mean age of (in years) was 13.02 ± 2.146. A self-administered structured questionnaire proforma was used. Pilot study was done to validate the questionnaire, which was constituted of nine items. The Student's t-test and ANOVA test along with stepwise multiple linear regression were applied for the statistical evaluation of means. The level of significance was set at 0.05. RESULTS: The overall awareness of orthodontist among the school going children was 45.1%. The knowledge of orthodontic procedures was significantly higher among girls (4.46 ± 1.671) when compared to boys (4.00 ± 1.489). When the results were compared according to the area of location most of the students in the urban areas gave a positive response regarding awareness when compared to children in the rural community. CONCLUSION: This group of preadolescents showed moderate level of awareness regarding orthodontic procedures as they mentioned that it helps in esthetics, better oral hygiene, mastication, and healthy lifestyle.

Hyaluronic acid receptor for endocytosis (HARE)-mediated endocytosis of hyaluronan, heparin, dermatan sulfate, and acetylated low density lipoprotein (AcLDL), but not chondroitin sulfate types A, C, D, or E, activates NF-κB-regulated gene expression.

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.

The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400-kDa, but not smaller or larger, hyaluronans.

The hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047-15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HA(TM) for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20-50 nM with an apparent K(m) ~10 nM, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.

The increased but non-predominant expression of Th17- and Th1-specific cytokines in Hashimoto’s thyroiditis but not in Graves’ disease

Hashimoto’s thyroiditis (HT) is considered to be mediated mainly by Th1 cells, but it is not known whether Graves’ disease (GD) is associated with Th1 or Th2 predominance. Th17 cells, a novel subset of Th cells, play a crucial role in the pathogenesis of various autoimmune disorders. In the present study, the expression of IL-17A and IFN-γ was investigated in patients with HT or GD. mRNA expression of IL-17A and IFN-γ in peripheral blood mononuclear cells (PBMC) from 43 patients with autoimmune thyroid disease (AITD) and in thyroid tissues from 40 AITD patients were measured by real-time qRT-PCR. The protein expression of IL-17A and IL-23p19 was examined by immunohistochemistry in thyroid tissues from 28 AITD patients. The mRNA levels of IL-17A and IFN-γ were higher in both PBMC and thyroid tissues of HT patients than in controls (mRNA levels are reported as the cytokine/β-actin ratio: IL-17 = 13.58- and 2.88-fold change and IFN-γ = 16.54- and 2.74-fold change, respectively, P < 0.05). Also, the mRNA levels of IL-17A and IFN-γ did not differ significantly in GD patients (P > 0.05). The high protein expression of IL-17A (IOD = 15.17 ± 4.8) and IL-23p19 (IOD = 16.84 ± 7.87) in HT was confirmed by immunohistochemistry (P < 0.05). The similar high levels of IL-17A and IFN-γ suggest a mixed response of Th17 and Th1 in HT, where both cells may play important roles in the destruction procedure by cell-mediated cytotoxicity.

The increased but non-predominant expression of Th17- and Th1-specific cytokines in Hashimoto's thyroiditis but not in Graves' disease.

Hashimoto's thyroiditis (HT) is considered to be mediated mainly by Th1 cells, but it is not known whether Graves' disease (GD) is associated with Th1 or Th2 predominance. Th17 cells, a novel subset of Th cells, play a crucial role in the pathogenesis of various autoimmune disorders. In the present study, the expression of IL-17A and IFN-γ was investigated in patients with HT or GD. mRNA expression of IL-17A and IFN-γ in peripheral blood mononuclear cells (PBMC) from 43 patients with autoimmune thyroid disease (AITD) and in thyroid tissues from 40 AITD patients were measured by real-time qRT-PCR. The protein expression of IL-17A and IL-23p19 was examined by immunohistochemistry in thyroid tissues from 28 AITD patients. The mRNA levels of IL-17A and IFN-γ were higher in both PBMC and thyroid tissues of HT patients than in controls (mRNA levels are reported as the cytokine/ß-actin ratio: IL-17 = 13.58- and 2.88-fold change and IFN-γ = 16.54- and 2.74-fold change, respectively, P < 0.05). Also, the mRNA levels of IL-17A and IFN-γ did not differ significantly in GD patients (P > 0.05). The high protein expression of IL-17A (IOD = 15.17 ± 4.8) and IL-23p19 (IOD = 16.84 ± 7.87) in HT was confirmed by immunohistochemistry (P < 0.05). The similar high levels of IL-17A and IFN-γ suggest a mixed response of Th17 and Th1 in HT, where both cells may play important roles in the destruction procedure by cell-mediated cytotoxicity.

CD40 C/T(-1) and CTLA-4 A/G(49) SNPs are associated with autoimmune thyroid diseases in the Chinese population.

This study was to investigate whether the common polymorphisms of CD40 and CTLA4 genes confer susceptibility to AITD in the Chinese population. A set of unrelated subjects including 303 GD patients, 208 HT patients, and 215 matched healthy controls were recruited. SNPs were genotyped by the method of PCR-RFLP. (1) As for CD40 C/T(-1) SNP, only a significant difference was found in allele frequencies between GD and control groups (P = 0.033). (2) On the part of CTLA-4 A/G(49) SNP, significant differences were found in genotype and allele frequencies between GD and control groups (P = 7.0 × 10(-5) and P = 0.002, respectively), and similar results were found between HT and control groups (P = 0.015 and P = 0.003, respectively). (3) The logistic regression analysis showed there was no interaction between CD40 and CTLA4 genotypes (P = 0.262). These results indicate that both CTLA-4 A/G(49) and CD40 C/T(-1) SNPs are associated with genetic susceptibility of GD, and CTLA-4 A/G(49) is also associated with HT.

N-Glycans on the link domain of human HARE/Stabilin-2 are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan.

The hyaluronic acid receptor for endocytosis (HARE)/Stabilin-2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ligand-binding sites are within the 190 kDa isoform, which contains approximately 25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N(2280), within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, we created human Flp-In 293 cell lines expressing membrane-bound or soluble ecto-domain variants of 190-HARE(N2280A). Membrane-bound HARE lacking Link domain N-glycans mediated rapid HA endocytosis, but purified 190-HARE(N2280A) ecto-domain showed little or no HA binding in ELISA-like, HA-HARE pull-down assays or by surface plasmon resonance analysis (which detected very high apparent affinity for 190-HARE ecto-domain binding to HA; K(d) = 5.2 nM). The results indicate that Link domain N-glycans stabilize interactions that facilitate HA binding to HARE.

The cytoplasmic domain of the hyaluronan receptor for endocytosis (HARE) contains multiple endocytic motifs targeting coated pit-mediated internalization.

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI(2485), FQHF(2495), NPLY(2519), and DPF(2534) (315-HARE numbering). Stably transfected cells expressing 190-HARE(DeltaYSYFRI), 190-HARE(DeltaFQHF), or 190-HARE(DeltaNPLY) (lacking Motifs 1, 2, or 3) had decreased (125)I-HA endocytosis rates of approximately 49, approximately 39, and approximately 56%, respectively (relative to wild type). In contrast, 190-HARE(DeltaDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only approximately 41%. Endocytosis was approximately 95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85-90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to approximately 7% of wild type. Tyr in NPLY(2519) is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE.