Aberrantly Glycosylated Research Articles

Does tonsillectomy have a role in the treatment of patients with immunoglobulin A nephropathy?

Immunoglobulin A nephropathy (IgAN) is an immune-complex mediated glomerulonephritis characterized by deposition of polymeric IgA (mainly IgA1) in the mesangium of the kidney [1]. It is the most common form of primary glomerulonephritis worldwide and is prevalent among all ages and racial demographics [2]. Its pathogenesis has been under investigation for the last several decades and one of the key players is the aberrant glycosylation pattern of IgA1. There is now convincing evidence that the hinge region of IgA1 heavy chain has defective glycosylation with a reduction in the galactose and or sialic acid residues [3]. As a result, the N-acetylgalactosamine in the IgA1 hinge region is exposed and recognized by IgG antibodies. This in turn leads to formation of IgG-IgA immune complexes and their deposition in the mesangium and renal injury that subsequently ensues [4–7]. It should be noted that aberrantly glycosylated IgA1 can also be seen in circulation in normal subjects following an immune response triggered by exposure to mucosal antigens such as food, bacteria or viruses. Thus, other factors, including genetic predisposition, are likely to influence the pathogenesis of IgAN [8]. The renal outcomes of IgAN vary significantly between individuals ranging from minimal proteinuria and stable renal function to development of end-stage renal disease (ESRD) in up to 50% of the cases [9]. Markers of poor prognosis include impaired kidney function at presentation, hypertension and persistent proteinuria >1 g/day in adults, and 0.5 g/day in children [10–12]. In addition, the recent Oxford classification has identified MEST (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis) as an independent renal biopsy indicator of poor renal outcome [13]. Treatments aiming to block the angiotensin II system, the use of corticosteroids, either alone or in combination with cytotoxic medications have been used in patients with IgAN with variable success [14–18]. In some patients, renal disease progresses despite treatment and thus a search for additional forms of therapy have been carried out including the use of tonsillectomy. What is the rationale behind performing tonsillectomy in patients with IgA nephropathy? First, recurrent tonsillitis is the most common extrarenal clinical manifestation in IgAN and there is a clear recognition that in some patients episodes of macroscopic hematuria and proteinuria are often associated with tonsillitis (synpharyngitic hematuria) [19, 20]. In fact, studies have shown that episodes of macroscopic hematuria can be reduced following tonsillectomy [21, 22]. Second, tonsils provide the first line of defense against inhaled pathogens. The foreign antigens, particularly bacterial polysaccharides, activate the Toll-like receptors (TLRs) of the cells of innate immunity, which ultimately results in activation of tonsillar B-cells and production of immunoglobulins particularly polymeric IgA1. Third, tonsils from patients with IgAN have an increased number of polymeric IgA1 compared with controls [23]. Forth, the polymeric IgA1 deposited in the kidney are, in part, of tonsillar origin. This was based on the evidence that IgA1 eluted from the mesangium of patients with IgAN specifically bound the nuclear regions of tonsillar cells obtained from the same patient and the binding was completely inhibited by the addition of antihuman IgA sera [24]. Fifth, abnormal IgAsecreting cells which predominantly produce polymeric IgA1 are found in the tonsillar tissue of patients with IgAN in contrast to IgG-secreting cells that predominate in tonsils of patients without IgAN [20, 25–27]. Taken together, it has been postulated that chronic tonsillitis in a susceptible patient may lead to increased production of aberrantly glycosylated IgA1, formation of IgG-IgA immune complexes and their deposition in the glomeruli triggering an inflammatory response [28]. As such, tonsillectomy has been suggested as a possible treatment modality in patients with IgAN. Several retrospective studies have evaluated the efficacy of tonsillectomy in treatment of IgAN with conflicting results as shown in Table 1 [21, 22, 29– 35]. Most of the studies, however, are from an era prior to IN F O C U S

Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover

Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient’s cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient’s cells, and its expression was considerably reduced, both in cells in vitro and in the patient’s epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.

Abnormal IgA1 O-Glycosylation in a Multi-ethnic population of IgA Nephropathy Patients in KwaZulu Natal, South Africa.

Abnormal IgA1 O-Glycosylation in a Multi-ethnic population of IgA Nephropathy Patients in KwaZulu Natal, South Africa.

Aberrant glycosylation of IgA in Wiskott-Aldrich syndrome and X-linked thrombocytopenia

Aberrant glycosylation of IgA in Wiskott-Aldrich syndrome and X-linked thrombocytopenia

PWE-011 Aberrantly glycosylated muc1 as a potential therapeutic target for Barrett's with high grade dysplasia and primary and metastatic oesophageal adenocarcinoma

IntroductionPatients with Barrett's (BE) associated oesophageal adenocarcinoma (OA), show strong expression of the mucin MUC1, but binding is not specific to dysplasia or cancer. Aberrant glycosylation of MUC1 (AG-MUC1) accompanies...

Su1882 Aberrantly Glycosylated MUC1 as a Potential Therapeutic Target for Barrett's With High Grade Dysplasia and Primary and Metastatic Oesophageal Adenocarcinoma

Introduction Patients with Barrett9s (BE) associated oesophageal adenocarcinoma (OA), show strong expression of the mucin MUC1, but binding is not specific to dysplasia or cancer. Aberrant glycosylation of MUC1 (AG-MUC1) accompanies the development of cancer in most epithelial tumours, exposing peptides hidden on normal cells. Humanised antihuman milk-fat globule-1 (Hu-HMFG1) antibody binds one of these regions. This study assesses expression of AG-MUC1 in the squamous-metaplasia-dysplasia-OA sequence, in OA specimens with infiltrated nodes and in cancer cell lines. Methods 34 paraffin embedded oesophageal tissue specimens were selected from patients with squamous (n=5), non-dysplastic BE (NDBE; n=3), low grade dysplasia (LGD; n=6), high grade dysplasia (HGD; n=9) and OA (n=11). 11 OA resection specimens with clear margins containing tumour and infiltrated nodes were also stained. Slides were immunostained with Hu-HMFG1 antibody and scored by an expert pathologist. Binding of HuHMFG1 to the cancer cell lines SKOV-3 (ovarian), MCF-7 (breast), OE-19, OE33 (oesophageal) and HT-29 (colon) was examined with flow cytometry using a secondary FITC conjugated antibody and analysed with FloJo. Results AG-MUC1 was significantly expressed (>33% positively stained cells) in 22% of HGD and 36% of OA specimens. Non-significant mild staining was seen in NDBE (100%), LGD (33%), HGD (44%) and OA (64%). In 27% of OA and 43% of HGD, adjacent squamous epithelium also stained. In surgically resected OA9s, 45% stained significantly for AG-MUC1 in primary tumour. Of these, 80% stained significantly in related lymph nodes. All OA resection margins were clear of significant staining. On flow cytometry, binding was noted on SKOV-3, MCF-7, OE-19 but not HT-29 or OE-33. Conclusion This pilot study demonstrates AG-MUC1 to be upregulated in the BE metaplasia-dysplasia-OA sequence with significant staining limited to HGD and cancer. Although some squamous staining was noted, this was likely a field effect as no significant staining was noted in OA resection margins. In patients with significantly stained primary tumour, most had significant staining in infiltrated lymph nodes. Finally, with flow cytometry we identified the OA cell line, OE-19 expressed AG-MUC1 in preparation for in vitro studies. AG-MUC1 targeting with the antibody Hu-HMFG1 offers a novel strategy to target HGD and OA, including those patients presenting with metastatic disease. Competing interests None declared.

The Gne M712T Mouse as a Model for Human Glomerulopathy

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.

Membrane-assisted online renaturation for automated microfluidic lectin blotting.

Aberrant glycosylation plays a pivotal role in a diverse set of diseases, including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multistage assay eliminates manual intervention steps required for slab-gel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and is completed using one instrument. The assay comprises non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by online post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments that trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein. Immunoglobulin A1 aberrantly glycosylated with galactose-deficient O-glycans was probed in ~6 min using ~3 μL of sample. This new microfluidic lectin blotting platform provides a rapid and automated assay for the assessment of aberrant glycosylation.

IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1

Aberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy. This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. Biosynthesis of these glycans occurs in a stepwise fashion beginning with the addition of N-acetylgalactosamine by the enzyme N-acetylgalactosaminyltransferase 2 and continuing with the addition of either galactose by beta1,3-galactosyltransferase or a terminal sialic acid by a N-acetylgalactosamine-specific alpha2,6-sialyltransferase. To identify the molecular basis for the aberrant IgA glycosylation, we established EBV-immortalized IgA1-producing cells from peripheral blood cells of patients with IgA nephropathy. The secreted IgA1 was mostly polymeric and had galactose-deficient O-linked glycans, characterized by a terminal or sialylated N-acetylgalactosamine. As controls, we showed that EBV-immortalized cells from patients with lupus nephritis and healthy individuals did not produce IgA with the defective galactosylation pattern. Analysis of the biosynthetic pathways in cloned EBV-immortalized cells from patients with IgA nephropathy indicated a decrease in beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity. Also, expression of beta1,3-galactosyltransferase was significantly lower, and that of N-acetylgalactosamine-specific alpha2,6-sialyltransferase was significantly higher than the expression of these genes in the control cells. Thus, our data suggest that premature sialylation likely contributes to the aberrant IgA1 glycosylation in IgA nephropathy and may represent a new therapeutic target.

Functional Proteomics Study Reveals That N-Acetylglucosaminyltransferase V Reinforces the Invasive/Metastatic Potential of Colon Cancer through Aberrant Glycosylation on Tissue Inhibitor of Metalloproteinase-

N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.

Melanophages reside in hypermelanotic, aberrantly glycosylated tumor areas and predict improved outcome in primary cutaneous malignant melanoma

Previously, hypermelanotic regions of cutaneous malignant melanoma (CMM) were found to contain a mixture of highly melanized melanoma cells and melanophages. Both cell types produced beta1,6-branched oligosaccharides. These sugars are used for motility by myeloid cells and cancer cells alike and are associated with poor survival in carcinomas of the breast, colon and lung. This study further investigated associations between melanophages and beta1,6-branched oligosaccharides and their potential contributions to patient outcome. Individual archival melanomas and high-throughput melanoma tissue microarrays were stained for melanophages with azure blue/S100 and for beta1,6-branched oligosaccharides with the lectin leukocytic phytohemagglutinin (LPHA, a selective marker for beta1,6-branched oligosaccharides). In primary CMM, melanophages were highly enriched in hypermelanotic, LPHA-positive tumor regions and correlated with improved outcome at 10- and 20-year follow ups. While the combination of melanophages, LPHA positivity and high pigmentation indicated better outcome, a subset of LPHA-positive cells not associated with melanophages indicated worse outcome. This is the first report of an anti-tumor role for the melanophage in melanoma biology. There appeared to be two classes of beta1,6-branched oligosaccharide-producing melanoma cells with opposing effects on outcome: one that attracted melanophages (better) and another that did not (worse). The findings disclose new aspects of the immune system and aberrant glycosylation in CMM.

Identification of target proteins of N‐acetylglucosaminyl transferase V in human colon cancer and implications of protein tyrosine phosphatase kappa in enhanced cancer cell migration

To gain a better understanding of the mechanism underlying colon cancer and to search for potential markers of colon cancer prognosis, a comparative proteomic analysis of colon cancer WiDr cells was conducted using 2-DE and lectin blot, followed by identification based on ESI-MS. Through these approaches 14 proteins were identified as candidate target proteins for N-acetylglucosaminyl transferase V (GnT-V) that would be expected to be implicated in the progression of colon cancer. We selected protein tyrosine phosphatase kappa (PTPkappa) as a model protein to validate this approach to the discovery of novel biomarkers in colon cancer. PTPkappa underwent an aberrant glycosylation in GnT-V-overexpressing WiDr cells, and the aberrantly glycosylated PTPkappa was vulnerable to proteolytic cleavage. The enhanced cleavage of PTPkappa in GnT-V-overexpressing cells was responsible for the mitigation of the homophilic binding capacity, resulting in an increase in cancer cell migration.

Alkaline phosphatase retained in HepG2 hepatocarcinoma cells vs. alkaline phosphatase released to culture medium: Difference of aberrant glycosylation

Liver tissue is the source of 90% of serum alkaline phosphatase (AP). The serum levels and structures of tumor marker proteins change under many disease conditions as well as cancer. The study was aimed at determining the type of alkaline phosphatase (AP) present in HepG2 hepatocellular carcinoma cell line. Alkaline phosphatase rich extracts of healthy human liver, HepG2 hepatocarcinoma cells, as well as the condition medium of HepG2 cells were prepared by extraction with 40% n-butanol and 30–50% acetone precipitation, and subjected to various chromatographic procedures. Lectin affinity chromatography of the samples with concanavalin A–Sepharose 4B showed considerable differences in the elution patterns. Non-denaturing polyacrylamide gel electrophoresis of the culture medium yielded a relatively slow migrating band of activity that coincided with none of the three bands of activity produced by the normal liver extract, nor with the bands of the cell pellet extract. Inhibition patterns were established by measuring the enzyme activities in the presence of varying concentrations of l-phenylalanine, l-leucine, l-homoarginine, and levamisole. The APs from the cell line were neuraminidase sensitive. According to the results the main AP produced and released to the medium by HepG2 cell line is an aberrantly glycosylated tissue non-specific AP. In addition, the differences between the cell-pellet AP and the culture medium AP seemed to stem from different sugar moieties in their structures.

Aberrant glycosylation in IgA nephropathy (IgAN)

Immunoglobulin A nephropathy (IgAN) patients exhibit circulating IgA1 with reduced galactose (Gal) and/or sialic acid (Neu5Ac) and increased exposure of N-acetylgalactosamine (GalNAc). These IgA glycoforms fix complement and in mesangial cells regulate integrin expression, enhance nitric oxide synthase (NOS) activity, decrease endothelial growth factor synthesis, meanwhile depressing proliferation and increasing apoptosis. Drugs can be targeted to the effects enhanced by aberrantly glycosylated IgA1 on mesangial cells. Recent data suggest that aberrant IgA1 glycosylation may modulate clinical expression and progression of IgAN.

Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy.

Transferrin receptor (TfR) has been identified as a candidate IgA1 receptor expressed on human mesangial cells (HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangial IgA1 deposits, and is overexpressed in patients with IgA nephropathy (IgAN). Here, structural requirements of IgA1 for its interaction with mesangial TfR were analyzed. Polymeric but not monomeric IgA1 interacted with TfR on cultured HMC and mediates internalization. IgA1 binding was significantly inhibited (>50%) by soluble forms of both TfR1 and TfR2, confirming that TfR serves as mesangial IgA1 receptor. Hypogalactosylated serum IgA1 from patients with IgAN bound TfR more efficiently than IgA1 from healthy individuals. Serum IgA immune complexes from patients with IgAN containing aberrantly glycosylated IgA1 bound more avidly to TfR than those from normal individuals. This binding was significantly inhibited by soluble TfR, highlighting the role of TfR in mesangial IgA1 deposition. For addressing the potential role of glycosylation sites in IgA1-TfR interaction, a variety of recombinant dimeric IgA1 molecules were used in binding studies on TfR with Daudi cells that express only TfR as IgA receptor. Deletion of either N- or O-linked glycosylation sites abrogated IgA1 binding to TfR, suggesting that sugars are essential for IgA1 binding. However, sialidase and beta-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR-IgA1 interaction as initial steps in IgAN pathogenesis.

Aberrant glycosylation modulates phosphorylation of tau by protein kinase A and dephosphorylation of tau by protein phosphatase 2A and 5

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in Alzheimer’s disease (AD). We recently found that the aberrant tau glycosylation precedes tau hyperphosphorylation in AD brain. In the present study, we developed assays to determine phosphorylation and dephosphorylation of tau at specific phosphorylation sites by using glycosylated tau purified from AD brain as a substrate. We then studied the effects of the aberrant glycosylation on phosphorylation and dephosphorylation of tau at each specific phosphorylation site. We found that deglycosylation of the aberrantly glycosylated tau decreased the subsequent phosphorylation of tau at Ser214, Ser262, and Ser356 in vitro by protein kinase A. On the other hand, deglycosylation of tau positively modulated the subsequent dephosphorylation by protein phosphatase 2A and protein phosphatase 5 in vitro at the phosphorylation sites Ser198, Ser199, and Ser202. Our results suggest that the aberrant glycosylation may modulate tau protein at a substrate level so that it is easier to be phosphorylated and more difficult to be dephosphorylated at some phosphorylation sites in AD brain. The combined impact of this modulation may be to make tau more susceptible to becoming abnormally hyperphosphorylated.

Alternative glycosylation of the insulin receptor prevents oligomerization and acquisition of insulin-dependent tyrosine kinase activity

Glucose deprivation leads to the synthesis of an aberrantly glycosylated (‘alternative’) and inefficiently processed form of the insulin proreceptor in 3T3-L1 adipocytes. To further explore the effect of aberrant (rather than absent) N-linked glycosylation of the insulin receptor, we examined the relationship of processing to function. Our studies show that the alternative form of the proreceptor does not oligomerize nor does it acquire the ability to undergo insulin-sensitive autophosphorylation. This along with an interaction with the glucose-regulated stress protein GRP78/BiP implies inappropriate folding/dimerization and retention in the ER. Glucose refeeding causes the post-translational modification of the alternative form of the proreceptor to a novel ‘intermediate’ form which is independent of new protein synthesis. As little as 100 μM glucose (or mannose) can induce this modification. In vitro digestion of the alternative and intermediate proreceptors with SPC1/furin shows that both the α- and β-subunit domains are glycosylated, albeit aberrantly. This implies that the aberrantly glycosylated proreceptor could serve as a substrate for SPC1 in a physiological setting if the receptor was able to interact with the enzyme in the appropriate compartment (i.e., the trans-Golgi network). Based on inhibitor studies, however, both the alternative and intermediate forms of the proreceptor appear to be primarily targeted to the proteasome for degradation.

An alpha2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas.

The MUC1 mucin is expressed on the luminal surface of most simple epithelial cells but in carcinomas, especially those of the breast and ovary, MUC1 is upregulated and aberrantly glycosylated. MUC1 contains a large amount of O-linked glycans which, in the mucin expressed by normal mammary epithelial cells, consist mainly of core 2 based structures carrying polylactosamine chains. However, the mucin expressed by breast carcinomas has shorter side-chains, often consisting of sialylated core 1 (Galbeta1-3GalNAc). in situ hybridization of primary breast tissue showed that a sialyltransferase (ST3Gal I), responsible for adding sialic acid to core 1 thereby terminating chain extension, is elevated in primary breast carcinomas when compared to normal or benign tissue. Furthermore, the level of mRNA expression encoding ST3Gal I is correlated to the intensity of staining seen with the antibody SM3, which specifically recognises underglycosylated, tumour associated MUC1. Thus, the aberrant glycosylation of MUC1 seen in breast carcinomas appears to be due, at least in part, to the elevation of ST3Gal I.

Mechanisms underlying aberrant glycosylation of MUC1 mucin in breast cancer cells.

The product of the MUC1 gene, the polymorphic epithelial mucin (PEM) is aberrantly glycosylated in breast and other carcinomas, resulting in exposure of normally cryptic peptide epitopes. PEM expressed by breast cancer cells contains more sialylated O-glycans and has a lower GlcNAc content than that expressed by normal cells. The exposure of peptide epitopes is thus thought to be due to the sugar side chains being shorter on the tumour-associated mucin. To investigate possible mechanisms underlying the different pattern of glycosylation in breast cancer cells, we analysed the pathways involved in the biosynthesis of O-glycan chains of mucins in normal and cancerous mammary epithelial cells. An immortalized mammary epithelial cells line originating from normal human milk. MTSV1-7, and three human breast cancer cell lines, BT20, MCF-7 and T47D, were studied. Glycosyltransferase activities assembling, elongating and terminating O-glycan core-1 [Gal beta 1-3GalNAc alpha-R] and core-2 [GlcNac beta 1-6 (Gal beta 1-3) GalNAc alpha-R] were present in the normal mammary cell line. Many of the glycosyltransferase activities were also expressed at variable levels in breast cancer cells. However, a sialyltransferase activity (CMP-sialic acid Gal beta 1-3GalNAc alpha 3-sialyltransferase) was increased several fold in all three cancer cell lines. Moreover, mammary cancer cell lines BT20 and T47D have lost the ability to synthesize core-2, as shown by the lack of UDP-GlcNAc: Gal beta 1-3GalNAc (GlcNAc to GalNAc) beta 6-GlcNAc-transferase activity, which corresponded to the absence of the mRNA transcript. However, MCF-7 breast cancer cells expressed this enzyme. Thus, the mechanism for the exposure of peptide epitopes in BT20 and T47D cells is proposed to be the loss of core-2 branching leading to shorter, sialylated O-glycan chains. A different mechanism is proposed for MCF-7 breast cancer cells.

S20.16 Characterisation of model systems for studying the aberrant glycosylation of the MUC1-1 gene product

The polymorphic epithelial mucin (PEM), coded for by the MUC1 gene is expressed on the luminal surface of many simple epithelial cells. Like other members of the mucin family, PEM contains a large domain which consists of tandemly repeated amino acids. This domain acts as a scaffold for the O-linked carbohydrate. PEM is upregulated in pregnant and lactating human mammary gland and in many adenocarcinomas. In addition, in malignant cells the molecule appears to be aberrantly glycosylated resulting in shorter sugar side-chains leading to the exposure of cryptic peptide epitopes such as that recognised by the antibody, SM3. In order to study the mechanisms underlying this aberrant glycosylation it is necessary to have an in vitro model system. To this end we have developed, by SV40 T antigen immortalisation, a cell line (MTSV1-7) which has many characteristics of normal mammary epithelial cells (Bartek et al., 1991, PNAS 88, 3520-3524). MTSV1-7 does not grow in soft agar or form tumours in nude mice and, unlike breast carcinoma lines, forms ordered structures in collagen gels (Berdichevsky and Taylor-Papadimitriou, 1991, Exp. Cell Res., 194, 267-274). Furthermore, by the criterion of antibody binding, MTSV1-7 appears to glycosylate the mucin like a normal cell. We are now in the process of purifying the MTSV1-7 mucin to confirm its carbohydrate composition by mass spectrometry (in collaboration with Professor Anne Dell). We intend to use this cell line and a breast carcinoma cell line to study the mechanisms responsible for the aberrant glycosylation of tumour cells.