Cancer-Type Expression of Tn Epitopes and LacdiNAc Structures: Human Cancer Cells Exhibit Distinctly Varying Levels of Heterogenous Ser/Thr Bearing Polypeptides, a Neutral β Galactosidase Converting T-Hapten to Tn, Ser/Thr: αGalNAc- and GlcNAc: β1-3/β1-4 GalNAc Transferase Activities

Research Article

Austin J Cancer Clin Res. 2021; 8(3): 1097.

Cancer-Type Expression of Tn Epitopes and LacdiNAc Structures: Human Cancer Cells Exhibit Distinctly Varying Levels of Heterogenous Ser/Thr Bearing Polypeptides, a Neutral β Galactosidase Converting T-Hapten to Tn, Ser/Thr: αGalNAc- and GlcNAc: β1-3/β1-4 GalNAc Transferase Activities

Chandrasekaran EV1*, Xue J1, Piskorz CF1, Locke RD1, Neelamegham S2 and Matta KL1,2*

1Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY, USA

2Department of Chemical and Biological Engineering, State University of New York, Buffalo, NY, USA

*Corresponding author: EV Chandrasekaran, Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

Matta KL, Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA; Department of Chemical and Biological Engineering, State University of New York, Buffalo, NY 14260, USA

Received: August 09, 2021; Accepted: September 13, 2021; Published: September 20, 2021


Terminal sugar-alteration in carbohydrate chains such as GalNAc replacing Gal in prostate and pancreatic cancers and Gal3-O-sulfation in breast, colon and gastric tumors could play a crucial role in cancer pathogenesis. We found the activities of cancer cell GalNAc transferases (GalNAc-Ts) as 0%, 0% <20%, 20-50% and 20-120% respectively towards Galβ1-3GalNAcα-OBn, 4-FGlcNAcβ1-6 (Galβ1-3)GalNAcα-O-Bn, LacNAcβ-O-Bn, GlcNAcβ1-4 GlcNAcβ-O-Bn and GlcNAcβ1-6GalNAcα-O-Bn as compared to GlcNAcβ-OBn. α-[6-³H]GalNAc-ylated endogenous cancer cells Ser/Thr polypeptides by the corresponding cancer cell αGalNAc-T were at variable levels, heterogenous, and exhibited complete binding to VVL-agarose and non-binding to WGA-, WFL- and ConA- agarose. PNA-agarose binding and non-binding radioactive products from [6-³H] GalNAc-ylated exogenous acceptor GlcNAcβ1-6(Galβ1-3) GalNAcα-O-Al indicated cancer type variable β1-3Galactosidase activities at neutral pH. TLC analysis identified two radioactive products by confirming PNA-agarose data. WGA-agarose tight binding and VVL-agarose weak binding respectively of the products [6-³H] GalNAcβ1-4 and β1-3GlcNAcβ-OBn isolated from the exogenous acceptor GlcNAc-β-O-Bn by Sep-Pak C18 method were used to quantitate β1-4 and β1- 3GalNAc-T activities in cancer cells. DU4475, MDA-MB-435S, PA-1, LNCaP, PC3, DU145, EG7 and GL261- OVA over-expressed β1-3GalNAc-T activity. Tumorigenic MDA-MB-435/LLC6 as compared to non-tumorigenic MDA-MB-435S contained ~2-fold each of αGalNAc-T and β1-4GalNAc-T. The breast cancer DU4475 uniquely expressed 10-fold β1-3GalNAc-T with respect to β1-4GalNAc-T. HPLC identified negligible β1-6GalNAc-T in cancer cells and high-level β1-3GalNAc-T in pancreatic and gastric tumors. It is known that Tn epitopes correlate with cancer progression and metastasis and β-galactosidase is a senescence-biomarker and moleculartarget for ovarian cancer. It is apparent that βGalNAc-T, Tn polypeptides, αGalNAc-T and neutral β1-3galactosidase could play a crucial role in cancer pathogenesis.

Keywords: Cancer cell O-glycans; Tn polypeptides; T-hydrolyzing neutral β1-3 galactosidase; αGalNAc- and β1-3/1-4GalNAc transferases; Lectinagarose; HPLC and TLC


AL: Allyl; Bn: Benzyl; BSA: Bovine Serum Albumin; ConA: Concavalin A; GalNAc-T: GalNAc Transferase; HPLC: High Performance Lipid Chromatography; NEU: Sialidase; PNA: Peanut Agglutinin; RM: Reaction Mixture; SA-β Gal: Senescence- Associated β Galactosidase; ST: Sialyltransferase; TLC: Thin Layer Chromatography; Tn GalNAcα-O-Ser/Thr: T Galβ1-3 GalNAcα-OSer/ Thr Type-I LDN GalNAcβ1-3GlcNAc (LacdiNAc) Type-II LDN GalNAcβ1-4GlcNAc (LacdiNAc); VVL: Vicia Villosa Lectin; WFL: Wisteria Floribunda Lectin; WGA: Wheat Germ Agglutinin


The glycoproteins containing complex glycan structures serve as the communication interface between cells and intracellular environment [1]. Several studies indicate that glycans and glycosylation of cellular proteins participate in the process of cancer cell adhesion, dissemination, and metastasis [2-5]. An unique expression of fucosyltransferase FT VI by colon cancer cell lines was identified by using GlcNAcβ1-4 GlcNAc as the specific acceptor [6]. The pattern of glycosyl- and glycan: Sulfotransferase activities in a wide range of human cancer cell lines was shown to be able to predict individual cancer associated signature carbohydrate structures [7]. A significant role for glycosyltransferases in invasion and intractability of pancreatic cancer became evident from a high-level overexpression of glycosyltransferases in pancreatic tumor [8]. Distinct changes in glycosyltransferases- specificities and lectin-binding by replacing terminal Gal with GalNAc in carbohydrate chains indicated that terminal sugar alteration could play a major role in cancer pathogenesis [8].

The LacdiNAc group is found mainly in N-glycans but also occurs in O-glycans [9-11]. The expression of the LacdiNAc group in N-glycans was reported to vary in human breast, prostate, ovarian and pancreatic cancers [12]. LacdiNAc-glycosylated PSA was better than the conventional PSA in identifying patients with clinically significant prostate cancer [13]. The NeuAcα2-6 GalNAcβ1-4GlcNAc sequence specifically found in secretory glycoproteins [14]. Recently mammalian glycoproteins were shown to carry GalNAcβ1-3GlcNAc on N-glycans in contrast to the presence of GalNAcβ1-4GlcNAc structures in N- and O- glycans of many mammalian glycoproteins, suggesting that GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc terminal units in glycans may have different roles in vivo [15].

The O-glycans impart unique features to mucin glycoproteins [16-19]. The first committed step in O-glycan biosynthesis is the addition of GalNAc to Ser/Thr [20]. Some αGalNAc-transferases such as T2 and T4 accomplish high-density glycosylation of certain protein substrates probably through binding as lectins [21]. Unsubstituted Tn epitopes occur in human cancers of colon, breast, bladder, prostate, liver, ovary and stomach and their presence correlate with cancer progression and metastases [22-24]. The expression of αGalNAc glycoconjugates detected by binding of HPA was found to be associated with metastatic competence and poor prognosis in a range of human adenocarcinomas [25]. ST6GalNAc1 mediated sialylation of Tn antigen and the frequent mutation of the cosmc chaperone that is required for the galactosyltransferase activity results in incomplete glycan structures [26].

The present study examined the acceptors-specificities of GalNAc transferases by using a variety of chemically synthesized compounds and identified by lectin-agarose affinity chromatography the levels of GlcNAc: β1-3 and β1- 4 GlaNAc tranferase activities in several human cancer cell lines. We found in these cell lines distinctly different levels of Tn epitope generating Ser/Thr containing small polypeptides (2-6 KD) and αGalNAc transferase activities. The present study found significant levels of a β galactosidase capable of converting T-glycotope to Tn at neutral pH in human cancer cell lines.

Materials and Methods

Cancer cell lines

T47D, MDA-MB-231, MCF-7, ZR-75-1, DU4475, MDA-MB- 435S, MDA-435/LCC6 (breast), COLO 205, SW1116, LS180 (colon), SW626, PA-1 (ovarian), HL60 (leukemic), Hep G2 (hepatic), LNCaP, PC3, DU145 (prostate), U87GB (glioblastoma), EG7 (lymphoma), RIF (fibrosarcoma) and GL261-OVA (glioma) were cultured as recommended by ATCC (Manassas, VA) and as reported in earlier studies [6,7,27]. All cell samples were homogenized with 0.1M Tris- Maleate pH 7.2 containing 2% Triton X-100 using a Dounce glass, hand-operated homogenizer. The homogenate was centrifuged at 16,000g for 1h at 4oC. Protein was measured on the supernatants by the BCA micro method (Pierce Chemical Co) with BSA as the standard. The supernatants were adjusted to 5mg protein/mL by adding the necessary amount of extraction buffer and then stored frozen at -20oC until use.

Tissue specimens

The tissue specimens were obtained from the tissue procurement facility of Roswell Park Cancer Institute. All tissue specimens were stored frozen at -70oC until processed as reported earlier [6,7,28,29). The tissue samples were homogenized at 4oC with 4 volumes (1ml/ per g tissue) of 0.1M Tris-Maleate pH 7.2 using Kinematica. After adjusting the concentration of Triton-X100 to 2%, these homogenates were mixed in the cold room for 1h using Speci-Mix (Thermolyne) and then centrifuged at 20,000g for 1h at 4oC. The clear fat free supernatant was adjusted to 10mg/ml protein by adding 0.1M Tris- Maleate pH 7.2 containing 2% TritonX100 and stored frozen at -20oC until use.

Acceptor compounds

The chemically synthesized compounds have already been used as acceptors for glycosyltransferases in our earlier studies and thus are well documented acceptor compounds for the study of glycosyltransferases [8,30-32].

Column chromatography

Biogel-P2 column or Biogel-P6 column (Fine Mesh; 1.0x116.0 cm) chromatography was carried out with 0.1 M pyridine acetate (pH5.4) as the eluent at room temperature. Void volume of this column is 30mL. The peak fraction containing [6-³H] GalNAc radioactivity were pooled, lyophilized to dryness, dissolved in a small volume of water and stored frozen at -20oC for further experimentation. Lectin-agarose affinity chromatography was carried out using columns of 7ml bed volume of ConA-, PNA-, WGA-, WFL- and VVL- agarose (Vector Lab, Burlingame, CA) under conditions recommended by supplier [6,32]. The radioactive sample was applied to the column in 1.0ml of the running buffer. After entry of the sample into the column bed, the sample remained in contact with the gel for 20min before starting elution with the running buffer. Fractions of 1ml were collected. The bound material from WGA-agarose was eluted with 0.5M GlcNAc and from WFL-agarose and VVL-agarose were eluted with 1.0M Gal. PNA-agarose and ConA agarose bound materials were eluted with 0.2M Gal and 0.1M methylα-D-mannoside respectively. Depending on the time of elution, the lectin binding interactions of WGA, VVL and WFL were classified into four categories; non-binding, weak binding, regular binding and tight binding as explained in our earlier report [32]. TLC was carried out on Silica gel GHLF (250μm scored 20X20cm; Analtech Newark DE). The solvent system 1-propanol/ NH4OH/H2O (12/2/5 v/v) was used [31]. The [6-³H] GalNAc products were located by scraping 0.5cm width segments of silica gel and soaking in 2.0ml water in vials followed by liquid scintillation counting. Pronase digestion of Biogel P-2 [6-³H] GalNAc containing peak 1 fraction was carried out in 600μl reaction mixture containing 4mg pronase CB, 0.1M Tris. HCL pH 7.0, 2mM CaCl2 and 2% ethanol at 37oC for 18h and then subjected to Biogel P-6 chromatography.

[6-3H] labelling of GalNAc β1-3GlcNAcβ-O-Bn, GalNAc β1- 4GlcNAcβ-O-Bn and GalNAc β1-6GlcNAcβ-O- Bn

These synthetic compounds (1.5μmol) were mixed separately with 20U of galactose oxidase and 200U of horse radish peroxidase in 0.1M Na-phosphate buffer pH 7 in 160μl reaction volume and incubated at 37oC for 21h and the oxidized GalNAc product was isolated by Sep- Pak method. The methanol eluates (5ml each) were concentrated to dryness and dissolved in 200μl of 0.05M Na-phosphate buffer pH 7, and then mixed with 100μl NaB [³H]4 (5mCi/500μl of 0.05M Naphosphate pH 7.0) and left at room temperature for 2h. Then 100μl NaBH4 (100mg/ml water) was added, mixed well intermittently, and left at room temp for 1h. Then these three solutions were neutralized by adding drops of acetic acid, left in the cold room overnight and the [6-³H] labelled compounds were isolated by Sep-Pak method.

Assay of GalNAc Transferases [8]

The incubation mixture (1.6ml) contained 0.1M Hepes pH 7.0 containing protease inhibitors (Calbiochem), exogenous synthetic acceptor Galβ1, 3 (GlcNAcβ1,6)GalNAc-o-Allyl (3.0μmol), 20mM Mn acetate, 7mM ATP, 3mM Na azide, UDP-GalNAc (Sigma Chemical Co. St. Louis, MO; 0.2μmol), UDP-(6-³H) GalNAc (American Radio Labeled Co. St. Louis, MO; 20μCi) and 1.0ml of Triton X100 solubilized cell extract. The final concentration of UDPGalNAc and the exogenous acceptor were 0.125mM and 1.9mM respectively. After incubation at 37oC for 20h, the incubation mixture was fractionated on a Biogel P2 column for the separation and quantitation of the radioactive products arising from endogenous and exogenous accetors. For the isolation of [6-³H] GalNAc-yl product from GlcNAcβ-O-Bn, 200μl of the incubation mixture contained 0.6μm of GlcNAcβ-O-Bn and 100μl of cell or tissue extract and other components in the same proportion as above. The radioactive products from benzylglycosides were separated by hydrophobic chromatography on a Sep-Pak C18 cartridge (Waters, Milford, MA) and elution of the product was done with 3mL methanol. The methanol eluate was concentrated to dryness by flash evaporation, dissolved in a small volume of water and stored frozen at -20oC for experimentation.


The HPLC separation [31] was performed on a C18 reverse-phase column using a gradient of acetonitrile in 10mM ammonium formate (pH 4.0). The sample injection volume was 20μl.


Lectin specificities

The present study utilized the specificities of PNA, VVL and WGA for characterizing N-acetyl galactosaminyl products from the endogenous and exogenous acceptors. Table 1 explains the specificities of these lectins using hapten inhibition assay showed that Galβ1-3GalNAcα-O-Al and its acrylamide copolymer are the most effective compounds for PNA. Antifreeze glycoprotein containing Galβ1-3GalNAcα-O-Ser/Thr chains was also highly effective. Any substitution on Gal in Galβ1-3GalNAcα- abolishes the inhibitory activity. The hapten inhibition of PNA binding is reduced considerably by methyl, sulfate or NeuAc substituent on C6-OH whereas they almost abolished the VVL binding. GalNAcα-O-Al and its acrylamide copolymer are effective in inhibiting VVL binding. Lectin agarose chromatography indicates NeuAcα2-3Galβ1-3 (GlcNAcβ1-6) GalNACα is the unit for regular binding of WGA whereas GalNAcβ1-4GlcNAc unit as such imparts tight binding to WGA. PNA-binding is inhibited by NeuAc and sulfate group on the β1-6 linked chain in mucin core 2 structure but not by αGal or Fuc.