Functional Consequences of Mannose and Asialoglycoprotein Receptor Ablation.

The mannose receptor (ManR, Mrc1) and asialoglycoprotein receptor (ASGR, Asgr1 and Asgr2) are highly abundant endocytic receptors expressed by sinusoidal endothelial cells and parenchymal cells in the liver, respectively. We genetically manipulated either receptor individually or in combination, revealing phenotypic changes in female and male mice associated with changes in circulating levels of many glycoproteins. Both receptors rise and fall in response to progesterone during pregnancy. Thirty percent of Asgr2(-/-) and 65% of Mrc1(-/-)Asgr2(-/-) mice are unable to initiate parturition at the end of pregnancy, whereas Mrc1(-/-) mice initiate normally. Twenty five percent of Mrc1(-/-)Asgr2(-/-) male mice develop priapism when mating due to thrombosis of the penile vein, but neither Mrc1(-/-) nor Asgr2(-/-) mice do so. The half-life for luteinizing hormone (LH) clearance increases in Mrc1(-/-) and Mrc1(-/-)Asgr2(-/-) mice but not in Asgr2(-/-) mice; however, LH and testosterone are elevated in all three knockouts. The ManR clears LH thus regulating testosterone production, whereas the ASGR appears to mediate clearance of an unidentified glycoprotein that increases LH levels. More than 40 circulating glycoproteins are elevated >3.0-fold in pregnant Mrc1(-/-)Asgr2(-/-) mice. Pregnancy-specific glycoprotein 23, undetectable in WT mice (<50 ng/ml plasma), reaches levels of 1-10 mg/ml in the plasma of Mrc1(-/-)Asgr2(-/-) and Asgr2(-/-) mice, indicating it is cleared by the ASGR. Elevation of multiple coagulation factors in Mrc1(-/-)Asgr2(-/-) mice may account for priapism seen in males. These male and female phenotypic changes underscore the key roles of the ManR and ASGR in controlling circulating levels of numerous glycoproteins critical for regulating reproductive hormones and blood coagulation.

The glycan-specific sulfotransferase (R77W)GalNAc-4-ST1 putatively responsible for peeling skin syndrome has normal properties consistent with a simple sequence polymorphisim.

Expanded access to DNA sequencing now fosters ready detection of site-specific human genome alterations whose actual significance requires in-depth functional study to rule in or out disease-causing mutations. This is a particular concern for genomic sequence differences in glycosyltransferases, whose implications are often difficult to assess. A recent whole-exome sequencing study identifies (c.229 C > T) in the GalNAc-4-ST1 glycosyltransferase (CHST8) as a disease-causing missense R77W mutation yielding the genodermatosis peeling skin syndrome (PSS) when homozygous. Cabral et al. (Genomics. 2012;99:202-208) cite this sequence change as reducing keratinocyte GalNAc-4-ST1 activity, thus decreasing glycosaminoglycan sulfation, as the mechanism for this blistering disorder. Such an identification could point toward potential clinical and/or prenatal diagnosis of a harmful medical condition. However, GalNAc-4-ST1 has minimal activity toward glycosaminoglycans, instead modifying terminal ß1,4-linked GalNAc on N- and O-linked oligosaccharides on specific glycoproteins. We find expression, processing and catalytic activity of GalNAc-4-ST1 completely equivalent between wild type and (R77W) sulfotransferases. Moreover, keratinocytes have little or no GalNAc-4-ST1 mRNA, indicating that they do not express GalNAc-4-ST1. In addition, loss-of-function of GalNAc-4-ST1 primarily presents as reproductive system aberrations rather than skin effects. These findings, an allele frequency of 0.004357, and a 10-fold difference in prevalence of CHST8 (c.299 C > T, R77W) across different ethnic groups, suggest that this sequence represents a "passenger" distributed polymorphism, a simple sequence variant form of the enzyme having normal activity, rather than a "driver" disease-causing mutation that accounts for PSS. This study presents an example for guiding biomedical research initiatives, as well as medical and personal/family perspectives, regarding newly-identified genomic sequence differences.

Modulation of mannose and asialoglycoprotein receptor expression determines glycoprotein hormone half-life at critical points in the reproductive cycle.

The rate at which glycoproteins are cleared from the circulation has a critical impact on their biologic activity in vivo. We have shown that clearance rates for glycoproteins such as luteinizing hormone (LH) that undergo regulated release into the circulation determine their potency. Two highly abundant, carbohydrate-specific, endocytic receptors, the asialoglycoprotein receptor (ASGR) and the mannose receptor (ManR) are expressed in the liver by parenchymal and sinusoidal endothelial cells, respectively. We demonstrate that the ManR mediates the clearance of glycoproteins such as LH that bear N-linked glycans terminating with ß1,4-linked GalNAc-4-SO4, as well as glycoproteins bearing glycans that terminate with Man. Steady state levels of mRNA encoding the ASGR and the ManR are regulated by progesterone in pregnant mice, reaching maximal levels on day 12.5 of pregnancy. Protein expression and glycan-specific binding activity also increase in the livers of pregnant mice. In contrast, ManR mRNA, but not ASGR mRNA, decreases in male mice at the time of sexual maturation. We show that levels of ManR and ASGR expression control the clearance rate for glycoproteins bearing recognized glycans. Thus, reduced expression of the ManR at the time of sexual maturation will increase the potency of LH in vivo, whereas increased expression during pregnancy will reduce LH potency until progesterone and receptor levels fall prior to parturition.

Molecular basis for protein-specific transfer of N-acetylgalactosamine to N-linked glycans by the glycosyltransferases ß1,4-N-acetylgalactosaminyl transferase 3 (ß4GalNAc-T3) and ß4GalNAc-T4.

Two closely related ß1,4-N-acetylgalactosaminyltransferases, ß4GalNAc-T3 and ß4GalNAc-T4, are thought to account for the protein-specific addition of ß1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of ß4GalNAc-T3 and ß4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by ß4GalNAc-T3 and ß4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mM MnCl(2); however, transfer is increasingly inhibited by concentrations of MnCl(2) above 1 mM and by anion concentrations above 15 mM. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-ß-p-nitrophenol (GlcNAcß-pNP) is not inhibited by concentrations of MnCl(2) or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. ß4GalNAc-T3 and ß4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC(50) for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nM for ß4GalNAc-T3 and 1.2 µM for ß4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.

Peptide-specific transfer of N-acetylgalactosamine to O-linked glycans by the glycosyltransferases ß1,4-N-acetylgalactosaminyl transferase 3 (ß4GalNAc-T3) and ß4GalNAc-T4.

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcß1,4GlcNAcß. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two ß1,4-N-acetylgalactosaminyltransferases, ß4GalNAc-T3 and ß4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in ß1,4-linkage to GlcNAc in vivo and in vitro. We now show that ß4GalNAc-T3 and ß4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to ß1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galß1,3(GalNAcß1,4GlcNAcß1,6)GalNAcαSer/Thr. The ß1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcß1,4GlcNAcß (LacdiNAc)-containing structures on specific glycoproteins.

Ablation of GalNAc-4-sulfotransferase-1 enhances reproduction by altering the carbohydrate structures of luteinizing hormone in mice.

Luteinizing hormone (LH), produced in the anterior lobe of the pituitary, is a member of the hypothalamic-pituitary-gonad axis that is required for production of the sex hormones estradiol, progesterone, and testosterone. Perturbations in levels of hormones associated with this axis can result in defects in sexual development and maturity. LH bears unique N-linked carbohydrate units that terminate with a sulfated N-acetylgalactosamine structure (GalNAc-4-SO(4)) that mediates its clearance from the blood. To determine the significance of this terminal structure, we ablated the gene encoding the sulfotransferase responsible for sulfate addition to GalNAc on LH, GalNAc-4-sulfotransferase-1 (GalNAc-4-ST1) in mice. Mice lacking GalNAc-4-ST1 exhibited increased levels of circulating LH. In male mice, this resulted in elevated levels of testosterone and precocious maturation of testis and seminal vesicles. Female mice lacking GalNAc-4-ST1 demonstrated elevated estrogen levels and exhibited precocious sexual maturation and increased fecundity. Female mice remained in estrus for prolonged periods and produced almost 50% more litters per mouse than wild-type mice over the same period of time. Thus, sulfate modification of the terminal glycosylation of LH plays a central role in regulating the hypothalamic-pituitary-gonad axis in vivo.

A necessary and sufficient determinant for protein-selective glycosylation in vivo.

A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with beta1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the beta1,4-N-acetylgalactosaminyltransferase (betaGT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned betaGTs, betaGT3 and betaGT4, that are able to transfer GalNAc to GlcNAc in beta1,4-linkage display the necessary glycoprotein specificity in vivo. Both betaGTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone alpha 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 betaGT3 and betaGT4 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 betaGT3 and betaGT4.

N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain.

Sorting protein-related receptor (SorLA/LR11) is a highly conserved mosaic receptor that is expressed by cells in a number of different tissues including principal cells of the collecting ducts in the kidney and neurons in the central and peripheral nervous systems. SorLA/LR11 has features that indicate it serves as a sorting receptor shuttling between the plasma membrane, endosomes, and the Golgi. We have found that a fraction of SorLA/LR11 that is synthesized in the kidney and the brain bears N-linked oligosaccharides that are modified with terminal beta1,4-linked GalNAc-4-SO(4). Oligosaccharides located in the vacuolar sorting (Vps) 10p domain (Vps10p domain) are modified with beta1,4-linked GalNAc when the Vps10p domain is expressed in cells along with either of two recently cloned protein-specific beta1,4GalNAc-transferases, GalNAcTIII and GalNAcTIV. Either of two sequences with basic amino acids located within the Vps10p domain is able to mediate recognition by these beta1,4GalNAc-transferases. The highly specific modification of oligosaccharides in the Vps10p domain of SorLA/LR11 with terminal GalNAc-4-SO(4) suggests that this unusual modification may modulate the interaction of SorLA/LR11 with proteins and influence their trafficking.

Spatial and temporal regulation of tenascin-R glycosylation in the cerebellum.

The cellular adhesion molecule tenascin-R is a multifunctional extracellular matrix component expressed exclusively in the central nervous system. The expression of tenascin-R by oligodendrocytes and small interneurons in the hippocampus and cerebellum is highly regulated during development of these regions. This complex glycoprotein displays both adhesive and anti-adhesive properties that contribute to the formation and maintenance of synapses. We have determined that tenascin-R associated with Purkinje cell bodies and their dendrites in the molecular layer of the cerebellum bears N-linked oligosaccharides terminating with beta1,4-linked GalNAc-4-SO(4), whereas tenascin-R in other regions of the cerebellum does not bear this modification. Expression of this unique sulfated carbohydrate structure is also temporally regulated, increasing throughout cerebellar development. The most dramatic increase in GalNAc-4-SO(4) occurs between postnatal days 14 and 21, corresponding to a period of Purkinje cell dendrite extension and synaptogenesis. The spatially and temporally regulated addition of this unique sulfated carbohydrate to tenascin-R may serve to modulate its adhesive/anti-adhesive or other biological properties in vivo.

Neuronal-specific synthesis and glycosylation of tenascin-R.

Tenascin-R (TN-R) is a member of the tenascin family of multidomain matrix glycoproteins that is expressed exclusively in the central nervous system by oligodendrocytes and small neurons during postnatal development and in the adult. TN-R contributes to the regulation of axon extension and regeneration, neurite formation and synaptogenesis, and neuronal growth and migration. TN-R can be modified with three distinct sulfated oligosaccharide structures: HNK-1 (SO(4)-3-GlcUAbeta1,3Galbeta1,4GlcNAc), GalNAc-4-SO(4), and chondroitin sulfate. We have determined that TN-R expressed in dendrite-rich regions of the rat cerebellum, hippocampus, and cerebral cortex is one of the major matrix glycoproteins that bears N-linked carbohydrates terminating with beta1,4-linked GalNAc-4-SO(4). The syntheses of these unique sulfated structures on TN-R are differentially regulated. Levels of HNK-1 on TN-R rise and fall in parallel to the levels of TN-R during postnatal development of the cerebellum. In contrast, levels of GalNAc-4-SO(4) are regulated independently from those of TN-R, rising late in cerebellar development and continuing into adulthood. As a result, the pattern of TN-R modification with distinct sulfated carbohydrate structures changes dramatically over the course of postnatal cerebellar development in the rat. Because TN-R interacts with a number of different matrix components and, depending on the circumstances, can either activate or inhibit neurite outgrowth, the highly regulated addition of these unique sulfated structures may modulate the adhesive properties of TN-R over the course of development and during synapse maintenance. In addition, the 160-kDa form of TN-R is particularly enriched for terminal GalNAc-4-SO(4) later in development and in the adult, suggesting additional levels of regulation.