Technical References

Structure and Function
The mucin family of glycoproteins is classified by the presence of tandem repeat structures rich in serines, threonines, and prolines that are extensively modified by Oglycosylation. The human MUC family consists of 20 members, of which are classified into subcategories based on whether they are secreted or membrane bound [1]. Secreted mucins (MUC-2, 3, 5AC, 5B, and 6) form a physical gel barrier that protects epithelial cells lining the respiratory and gastrointestinal tracts and ductal surfaces of specialized organs such as the pancreas, kidney, and liver. Membrane bound mucins (MUC-1, 3, 4, 12, 13, 16, and 17) also contribute to the formation of a protective mucous gel through ectodomains of O-glycosylated tandem repeats that extend from the apical surface of the cell. Membrane bound mucins typically contain a sea urchin sperm protein, enterokinase and agrin (SEA) domain that resides between the glycosylated ectodomain and the transmembrane domain (Figure 1). Autoproteolysis of the MUC-1 SEA domain results in the formation of a stable non-covalent dimer, consisting of the N-terminal ectodomain and a C-terminal transmembrane subunit [2-4].

Figure 1. MUC-1 Overview The mucin family of glycoproteins is characterized by their tandem repeat domain, which undergoes extensive Olinked glycosylation following translation (italicized in RED). MUC-1 is cleaved by autoproteolysis and forms a stable non-covalent dimer, consisting of a small cytoplasmic tail and a large N-terminal ectodomain. Following insertion into the plasma membrane, the N-terminal ectodomain is shed into circulating blood, while the C-terminal tail returns to the cytoplasm where it is phosphorylated, thus activating signal transduction pathways. At Bioprocessing, Inc., we purify carbohydrate antigens that are presented on the surface of the MUC-1 molecule. These epitopes are considered Tumor Antigens based on their increased frequency detected in cancer patients circulating fluids.

MUC-1 localizes to the apical border of normal epithelial cells [5]. Transformation and a loss of polarity in epithelial cells results in MUC-1 expression over the entire surface of the cell, as seen in Figure 2 [5].

Figure 2. ZR-75-1 Immunohistochemistry Immunohisochemistry was performed on ZR-75-1 cells. Cells were washed and resuspended in 1X PBS followed by fixation in 4% paraformaldehyde. Fixed cells were permeabilized followed by staining with our MUC-1 specific monoclonal antibody (6A4 catalogue #2250-8215) that is specific for the MUC-1 antigen Ca15-3.

Furthermore, overexpression of MUC-1 causes transformation [6] and resistance to stress induced apoptosis in normal epithelial cells [7-12]. The MUC-1 N-terminal ectodomain (MUC1-N) contains variable numbers of highly glycosylated 20 amino acid tandem repeats, that have a high density of O-linked oligosaccharide residues (Ser/Thr), and is shed into circulating blood. The shed form of MUC-1 has been reported to have a molecular weight of 330-400 kD [13-15].

There have been three variant isoforms of MUC-1 identified, MUC-1X, Y, or Z, that contain variable amounts of carbohydrate content ranging from 50-80% [16, 17]. MUC-1 isolated from normal epithelium contains less carbohydrate by having shorter chains of galactose N-acetyl glucosamine and N-acetyl galactosamine than in tumor epithelium. Carbohydrates are added to MUC-1 by core 2 O-glycans of sialyl lewis x structure (NeuAc2-3GalB1-4 (Fuc1-3) GlcNAc-R (SLex)) through post-translational modifications. In tumor cell lines and serum collected from patients with cancer, the sialylated lewis A moiety can be part of the core structure of MUC-1, which is detected by using a monoclonal antibody for sialylated lewis A (Ca19-9) [18-20]. Additionally, MUC-1 has been shown to be autoproteolytically cleaved from a 110-residue SEA transmembrane domain on tumor cell surfaces, which is also part of the Ca125 molecule expressed in ovarian cancer [4, 21, 22].

Purity Ratios
At Bioprocessing, Inc., we isolate breast tumor antigen (Ca15-3) from natural product and cell culture supernatant collected from human breast adenocarcinoma cells, obtaining average ratios of 2.5 X 104 Units/mg using optical density (OD) at 280 nm. Ca15-3 immunoblots at ~400 kD using a Ca27.29 monoclonal antibody (Figure 4). Additionally, we have observed that Br antigen forms aggregates that migrate at >400 kD when placed over a size exclusion column. Currently, a standard calculation to determine the purity ratio of Ca15-3 remains to be established. Purified Ca15-3 contains less carbohydrate and a significantly higher amount of protein, whereas Ca19-9 has a greater content of carbohydrate as compared with protein. From this, it is estimated that there are 5.9 ng of available Ca15-3 antigen per 1000 U (1 KU) of MUC-1. Based on this, 1 mg of highly purified MUC-1 is 25-50 KU and contains 0.15-0.30 ug of Ca15-3 available antibody reactive determinants.

Analysis
We employ PAGE analysis to determine Ca15-3 purity for both natural product and cell culture supernatant. We load 1-2 KU (5-10 ug) on a 4-20% PAGE gel run under reducing conditions followed by staining with GelCode Blue (Thermo Fisher Scientific Inc., Rockford, IL) (Figure 3).

Figure 3. PAGE Analysis of Pure Ca15-3 Antigen In order to determine the purity of Ca15-3 antigen that is purified using size exclusion chromatography, we subject 250 U (catalogue #2250-2301) to PAGE analysis. Samples are run on a 4-20% PAGE gel under reducing conditions and stained with GelCode Blue. Results demonstrate the relative purity of Ca15-3.

Immunoblotting for purified Ca15-3 using Ca27.29 under reducing conditions on a 4-20% gradient PAGE gel (Bio-Rad, Hercules, CA) displays a double band at ~400 kD (Figure 4). As seen in Figure 4, a faint double band appears as a result of posttranslational modifications of MUC-1, which is recognized by Ca27.29.

This large glycoprotein is defined by the carbohydrate epitope Ca19-9 located on MUC-1, and is referred to as sialylated lewis A (Lea). It is expressed by epithelial tumors of the gastrointestinal (GI) tract including the stomach, small and large intestines, colon, pancreas, and cervix. Ca19-9 mucin circulates in the blood and accumulates in ascites or pleural fluid of patients with GI cancer. Using a RIA kit specific for the Ca19- 9 epitope (Centocor/Fujirebio), 1 Unit of Ca19-9 corresponds to 0.59 ng/mL of the 116NS-19-9 antibody reactive determinants. This corresponds to ~1.7 U/ng of total protein or 1 KU equals 0.59 ug of Ca19-9 antigen. Other antibodies have been produced that recognize different determinants on the same molecule, including Ca50, Span-1, Dupan-2, and SSEA-1, which are measured in terms of each specific antibodies reactive epitope units [23, 24].

Figure 4. Western Blot of Pure Ca15-3 Antigen Purified Ca15-3 (250 U of catalogue #2250-2301) was subjected to western blotting with Ca27.29, a monoclonal antibody that is specific for Ca15-3 epitope on MUC-1. Results demonstrate the purity in addition to the degree of glycosylation, as seen by the smearing pattern on the blot.

Purity Ratios
Reported Ca19-9 antigen purity ratios are ~2.5 X 106 U/mg of total protein [25, 26]. Ca19-9 purified from natural product is first precipitated with perchloric acid and then purified over a size exchange column. We consistently obtain Ca19-9 antigen purity ratios from natural product ranging from 2.0-5.0 X 106 U/mg, as measured by OD 280 nm. This range represents the variability from patient to patient on available Ca19-9 antigen. Given that 1 KU of Ca19-9 equals 0.59 ug of antibody reactive determinants means that 2,000 to 5,000 KU of pure Ca19-9 is 1-3 mgs of reactive antibody determinants per mg by OD 280 nm. For cell culture supernatant, we use SW116 colon cancer cells, which secrete Ca19-9. The cell culture conditioned media is collected, concentrated, and subjected to size exclusion chromatography. Because we prepare the natural product and cell culture supernatant differently prior to purification, we also obtain differences in relative purity ratios.

Analysis
We employ PAGE analysis to determine Ca19-9 purity for both natural product and cell culture supernatant. Loading 1-2 KU (0.5-1 ug) followed by staining with GelCode Blue (Thermo Fisher Scientific Inc., Rockford, IL). It is difficult to detect Ca19-9 based on the aggregates that it forms when in a concentrated solution. Because we purify MUC-1 antigen using size exclusion chromatography, there are some contaminants that appear at lower molecular weights, which are mostly albumin and acidic serum proteins that are bound to MUC-1.