Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)␣2,6GalNAc␤1,4GlcNAc␤1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Sia␣2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Sia␣2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Sia␣2,6Gal␤1,4GlcNAc but not those with the terminal Sia␣2,3Gal␤1,4GlcNAc. We propose that glycoproteins bearing terminals Sia␣2,6GalNAc and Sia␣2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing ␣2,6-linked Sia.clearance ͉ galactose ͉ N-acetylgalactosamine ͉ hepatic lectin ͉ serum glycoproteins

Background: Unique glycan structures on glycoprotein hormones are recognized by glycan-specific receptors that control hormone half-life in the blood. Results: Clearance rates for recognized glycoproteins are determined by the level of receptor expression in the liver. Conclusion: Expression of glycan-specific receptors is modulated at critical points in the reproductive cycle. Significance: Glycans and glycan-specific receptors play critical roles in reproduction.

IntroductionOvulation, fertilization, and implantation are complex processes that depend on the exquisitely coordinated interaction of neurons in the hypothalamus, gonadotroph cells in the anterior pituitary, and target cells in the ovary. These three participants form the gonad/pituitary/hypothalamic axis. Abnormalities in the regulation of this axis can result in infertility and miscarriage (1-3). Peptide-releasing factors produced in the hypothalamus stimulate gonadotroph cells to release the glycoprotein hormones follitropin (FSH) and lutropin (LH) into the circulation. FSH directs follicular development and oocyte maturation, whereas LH is responsible for the rupture of antral follicles to release the ovum and for the conversion of postovulatory follicles into corpora lutea (4). The precisely controlled production of estrogen and progesterone, initially by developing follicles and subsequently by corpora lutea, is essential for differentiation of the uterus into a receptive state for the fertilized egg and for implantation (5-9).LH characteristically demonstrates an episodic rise and fall in the circulation that is critical for the biologic activity of LH in vivo. Gonadotropin-releasing hormonestimulated release of LH from dense core granules in pituitary gonadotrophs generates these pulses at specific intervals. The amplitude and frequency of LH pulses produced in the circulation are in turn regulated by progesterone and estrogen levels (10-14). The pulsatile rise and fall in circulating LH levels results in the episodic stimulation of the LH receptor, a G protein-coupled receptor (15). Episodic stimulation may be critical because the activated form of the LH receptor is recognized by β-arrestin, resulting in uncoupling from adenylyl cyclase and internalization of the occupied receptor (16). Replenishment at the cell surface with unoccupied LH receptor requires periods between pulses when the cells are not exposed to the hormone.The episodic rise and fall in LH levels also requires that LH have a short half-life in the circulation. The circulatory half-life of LH, but not FSH, is precisely controlled by the Man/GalNAc-4-SO 4 receptor that we have identified in hepatic endothelial cells (17,18). We initially demonstrated that LH bears unique oligosaccharide structures that terminate with β1,4-linked GalNAc-4-SO 4 whereas FSH bears structures that terminate with sialic acid linked to galactose (19-21). We have shown that it is the cysteine-rich domain located at the aminoterminus of the Man/GalNAc-4-SO 4 receptor, expressed at high levels in hepatic endothelial cells, that specifically binds terminal β1,4-linked GalNAc-4-SO 4 located on the N-linked oligosaccharides of LH (22-24). This binding mediates the rapid clearance of LH from the circulation. The Man/GalNAc-4-SO 4 receptor expressed by hepatic endothelial cells is present in the form of a dimer that must simultaneously bind to two terminal GalNAc-4-SO 4 moieties on LH to achieve the apparent K d of 1.6 × 10 -7 M we have observed with isolated cells (2...

A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with ␤1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the ␤1,4-N-acetylgalactosaminyltransferase (␤GT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned ␤GTs, ␤GT3 and ␤GT4, that are able to transfer GalNAc to GlcNAc in ␤1,4-linkage display the necessary glycoprotein specificity in vivo. Both ␤GTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone ␣ subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxyl-terminal 19-amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. The addition of this 19-amino acid sequence to the carboxyl terminus of Trf confers full acceptor activity onto Trf for both ␤GT3 and ␤GT4 in vivo. The complete 19-amino acid sequence is required for optimal GalNAc addition in vivo, indicating that the peptide sequence is both necessary and sufficient for recognition by ␤GT3 and ␤GT4.

We have developed a deep-scale proteome and phosphoproteome database from 44 representative Acute Myeloid Leukemia (AML) patients from the LAML TCGA dataset, and 6 healthy bone marrow derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of post-transcriptionally regulated proteins both globally (i.e. in all AML samples), and also, in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2‑oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in mRNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with post‑transcriptionally increased protein abundance, and showed that they interact with NPMc, but not wildtype NPM1. We identified two cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies, and confirmed these targets via flow cytometry. Finally, we detected nearly 30,000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1 and PRKCD. FLT3‑TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK, and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis.