Functional Significance of Glycoprotein Clearance by the Asialoglycoprotein Receptor and the Mannose/GalNAc-4-SO4 Receptor

Glycosylation plays an important role in many biological functions. Two highly abundant, carbohydrate-specific, endocytic receptors reside in parenchymal and endothelial cells of the liver. Our lab has shown that the asialoglycoprotein receptor: ASGR) is capable of clearing glycoproteins bearing terminal Siaalpha2,6GalNAc as well as ones bearing terminal Gal/ GalNAc and that the Mannose/GalNAc-4-SO4 receptor: MR) is capable of clearing glycoproteins bearing terminal GalNAc-4-SO4. I am taking a genetic approach identifying endogenous ligands for the ASGR and the MR in vivo and establishing the biologic significance of clearing these glycoproteins from the blood. A number of glycosylated hormones such as luteinizing hormone: LH), thyroid stimulating hormone, and the prolactin like proteins bear structures that would be recognized by either the ASGR or the MR and clearance would potentially help regulate their concentrations following release into the blood. I have obtained ASGR-/-, MR-/-, and ASGR-/-MR-/- mice. I am using mass spectrometric methods to identify glycoproteins that are elevated in the blood of these mice. Glycoproteins bearing Siaalpha2,6Gal are elevated in ASGR-/- mice suggesting that glycoproteins with Siaalpha2,6Gal rather than terminal Gal or GalNAc are cleared by the ASGR. Many are acute phase proteins and we propose that the ASGR helps regulate their relative concentrations in vivo and enhances their increase during the acute phase response. LH bears terminal GalNAc-4-SO4 and the half life of LH is increased in MR-/- mice indicating that the MR does account for LH clearance in vivo. ASGR-/- mice also have elevated LH, but the half life is not increased indicating an alternative mechanism of elevating LH in ASGR-/-, likely through a protein bearing Siaalpha2,6Gal/GalNAc. Ablation of both the MR and ASGR results in mice that are fertile, but unable to induce parturition. This suggests clearance of proteins bearing Siaalpha2,6Gal/GalNAc and GalNAc-4-SO4 is critical for appropriate plasma protein levels changes associated with parturition. Clearance by the ASGR and MR may contribute to regulating the concentrations of a range of glycoproteins including acute phase proteins and hormones

An Antibody to De-N-Acetyl Sialic Acid Containing-Polysialic Acid Identifies an Intracellular Antigen and Induces Apoptosis in Human Cancer Cell Lines

Polysialic acid (PSA), an α2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac), is developmentally regulated and its expression is thought to be restricted to a few tissues in adults. Recently, we showed that two human pathogens expressed a derivative of PSA containing de-N-acetyl sialic acid residues (NeuPSA). Here we show that an epitope identified by the anti-NeuPSA monoclonal antibody, SEAM 3 (SEAM 3-reactive antigen or S3RA), is expressed in human melanomas, and also intracellularly in a human melanoma cell line (SK-MEL-28), a human T cell leukemia cell line (Jurkat), and two neuroblastoma cell lines (CHP-134 and SH-SY5Y). SEAM 3 binding induced apoptosis in the four cell lines tested. The unusual intracellular distribution of S3RA was similar to that described for the PSA polysialyltransferases, STX and PST, which are also expressed in the four cell lines used here. Interestingly, suppression of PST mRNA expression by transfection of SK-MEL-28 cells with PST-specific short interfering RNA (siRNA) resulted in decreased SEAM 3 binding. The results suggest further studies of the utility of antibodies such as SEAM 3 as therapeutic agents for certain malignancies

Immuno-fluorescence analysis of SEAM 3 binding to human SK-MEL-28 melanoma and CHP-134 neuroblastoma cells.

<p>Light micrographs of SK-MEL-28 and CHP-134 show the general shape of cells with nuclear DNA indicated in blue. Anti-NeuPSA mAb SEAM 3 binding (in red) to SK-MEL-28 and CHP-134 cells not treated or treated with Triton X-100 to permeablize the cells, was compared to anti-GD3 and –PSA in green, as indicated. Subcellular localization of S3RA in SK-MEL-28 cells with Golgi (giantin, golgin 97 and Tuba) and ER (calnexin) markers are shown in the bottom panel in yellow. Arrows indicate granular vesicular-like structures with relatively intense SEAM 3 staining. Reference bars = 20 µm.</p

Intracellular localization of de-N-acetyl PSA in melanoma and neuroblastoma cell lines.

<p>SK-MEL-28 and SH-SY5Y cells were either untreated to detect surface binding (upper panel in each set of two panels), or treated with Triton-X 100 to detect intracellular binding (lower panel) by flow cytometry. Cells were incubated with 5 µg/ml of each primary antibody, followed by incubation with 2 µg/ml Alexa Fluor 488-conjugated secondary antibody. Irrelevant murine IgG2b, IgG3, and IgG1 mAbs were used as negative controls for SEAM 3, anti-GD3, and anti-NCAM, respectively, and were used to determine baseline fluorescence. Binding was detected using Guava EasyCyte flow cytometer. Gates used to define cells positive for binding are indicated at the top of each histogram.</p

Effect of polysialyltransferase PST siRNA on SEAM 3 binding to SK-MEL-28 cells.

a<p>Binding to Triton X-100-treated SK-MEL-28 cells 72 hours after transfection with the indicated siRNA was measured by flow cytometry as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027249#pone-0027249-g003" target="_blank">Figure 3</a>.</p>b<p>P values comparing the mean percentages of SEAM 3-positive cells were determined using an unpaired two tailed t test.</p

SEAM 3 mediates antibody dependent cytotoxicity by inducting apoptosis.

<p>(A), Antibody dependent cytotoxicity of SEAM 3 against SK-MEL-28, CHP-134, SH-SY5Y, and Jurkat cells as measured by LDH release assay. Each cell line was incubated with increasing concentrations of SEAM 3 for 16 hrs. LDH release was measured and percent cytotoxicity was determined using spontaneous release and maximal release following treatment with Triton X-100. (B), Analysis of SEAM 3 mediated apoptosis against SK-MEL-28 melanoma cells by flow cytometry. SK-MEL-28 cells were incubated with an irrelevant IgG2b mAb (5 µg/ml), DMSO, 0.1 µM Staurosporine, or 5 µg/ml SEAM 3 for 12 or 24 hours. Cells were then stained with fluorescently labeled annexin V and propidium iodide and the fraction of live (open bars), apoptotic (cross-hatched bars), and dead cells (black bars) was measured by flow cytometry.</p

Relative quantification of PST mRNA in SK-MEL-28 cells treated with scrambled siRNA or PST-specific siRNA.

<p>SK-MEL-28 cells were transiently transfected with 50 nM siRNA for 72 hours in triplicate, then total RNA from each cell line was reverse-transcribed into cDNA and used as the template in the qRT-PCR reaction. Relative quantification was determined using the comparative CT method, normalized to GAPDH mRNA.</p

Expression of PSA-NCAM and GD3 in human cancer cell lines tested.

a<p>Recent studies have suggested that Jurkat cells may transiently express GD3 during Fas (CD95)-signaling <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027249#pone.0027249-Kang1" target="_blank">[44]</a> and normal human leukocytes have been shown to express PSA-NCAM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027249#pone.0027249-Drake1" target="_blank">[10]</a>.</p

Summary of SEAM 3 binding to cancer cell lines determined by flow cytometry.

<p>Summary of SEAM 3 binding to cancer cell lines determined by flow cytometry.</p