Glycoprotein methods protocols - biotechnology 048-9-471-486.pdf

Glycoprotein methods protocols - biotechnology

39

Expression of MUC1 in Insect Cells

Using Recombinant Baculovirus

Pawel Ciborowski and Olivera J Finn

1 Introduction

MUC1 mucin undergoes multistep posttranslational modifications before it is finally expressed on the apical surface of mammalian ductal epithelial cells Two early

precursor proteins are both N-glycosylated and differ in molecular weight owing to a

proteolytic cleavage of a 20-kDa fragment Proteolytically modified form is

trans-ported to the Golgi, where it undergoes extensive, although not complete,

O-gly-cosylation on serine and threonine residues within the tandem repeat (TR) region MUC1 is then transported to the cell surface For additional glycosylation and

sialylation, surface MUC1 is internalized and directed to trans-Golgi compartments.

Mature form is again transported to the cell surface (1).

MUC1 expressed by malignant epithelial cells such as breast and pancreatic adeno-carcinomas is underglycosylated (aberrantly glycosylated), which makes it

structur-ally and antigenicstructur-ally distinct from that expressed by normal cells (2) As such, it may

be an excellent target for immunotherapy One of the ways to utilize tumor-specific forms of this molecule is as immunogens Purifying these forms from tumor cells is not feasible because it is a labor-intensive process that gives low yields A much more desirable approach is purification of a recombinant molecule from an appropriate expression system Recombinant MUC1 expressed in a convenient prokaryotic

sys-tem that does not glycosylate proteins, such as Escherichia coli, undergoes rapid and

random proteolytic degradation To obtain underglycosylated recombinant tumorlike forms of MUC1 in mammalian cells through expression vectors such as vaccinia virus, retroviral vectors, and plasmid vectors requires a prolonged treatment of infected or

transfected cells with toxic and expensive inhibitors of O-linked glycosylation (3,4).

Furthermore, vaccinia and retroviral constructs spontaneously recombine out most TRs

that characterize the major portion and the most immunogenic portion of MUC1 (5).

We explored the baculovirus system that allows expression of MUC1 mucin in

Spodoptera frugiperda Clone 9 (Sf-9) insect cells We found that these cells, when

From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

infected with a MUC1 recombinant baculovirus, produce fully glycosylated, full-size

(no deletion of TRs) molecules that are expressed on the cell surface (6) Moreover,

under specific starvation growth conditions that we determined empirically, Sf-9 cells can also produce underglycosylated MUC1, similar to the MUC1 produced by tumor cells The state of glycosylation of various forms can be evaluated by their migration

in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and

reactivity with different anti-MUC1 antibodies in Western blot analysis (6,7) In this

chapter, we present the techniques of expression of MUC1 mucin using three

baculoviral vectors: pBlueBacIII, pFastBac, and pIE1-4 Additional vectors are

com-mercially available and, as one can expect, more will emerge on the market in the future In our opinion, they provide an ideal expression system to study different forms

of MUC1 protein, their function, and utility.

2 Materials

2.1 Cloning Reagents

1 Vectors: pBlueBacIII was purchased from Invitrogen, San Diego, CA (see Note 1);

pFastBac was purchased from Gibco, Life Technologies, Grand Island, NY; and pIE1-4 was purchased from Novagen, Madison, WI

2 Competent E coli cells such as MAX Efficiency DH5α™ Competent Cells and MAX Efficiency DH10Bac™ Competent Cells were obtained from Gibco-BRL

3 Restriction enzymes, agarose, ligase, and other reagents for cloning may be obtained from any supplier of molecular biology reagents Wizard™ Minipreps and Wizard™ Megapreps were obtained from Promega, Madison, WI Cationic liposomes InsecticinPlus™ were obtained from Invitrogen, but can be also obtained from other commercial sources BluoGal and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sigma,

St Louis, MO; X-gal was purchased from Boehringer Mannheim, Indianapolis, IN; and SeaPlaque agarose was purchased from FMC BioProducts, Rockland, ME

2.2 Cells, Media, and Antibodies

1 The insect cell line Sf-9 can be obtained from American Type Culture Collection (Rockville, MD) or from other suppliers such as Invitrogen, San Diego, CA

2 Hink’s TNM-FH Insect Medium can be obtained from several sources such as JRH Bio-sciences, Lenexa, KS Penicillin, streptomycin, fungizone, and geneticin can be obtained from Gibco Fetal bovine serum (FBS) was from Gibco-BRL

3 Anti-MUC1 antibodies used in this study are not commercially available Monoclonal antibodies (MAbs) used for Western blot and flow cytometry analysis are listed in

Table 1 The TD-4 MUC1 Workshop (see ref 7, pp 1–152) provides the most up-to-date

list of anti-MUC1 antibodies, their specific reactivities, and their sources

4 Tissue culture flasks, plates, roller bottles, and disposable plastic tubes of various sizes can be obtained from various sources, e.g., Sarsted, Falcon, etc Any 27°C incubator can

be used, although one with a water jacket is recommended

2.3 Western Blot

All reagents and equipment for PAGE and Western blot, except nitrocellulose, were purchased from Bio-Rad, Hercules, CA Other suppliers can also be used Nitrocellu-lose BioBlot-NC was purchased from Corning Costar, Corning, NY

Chemilumines-cence Western blotting detection kit was purchased from Amersham, Buckinghamshire, England.

3 Methods

3.1 Vector Construction

The cDNAs coding for MUC1 of various lengths owing to various numbers of TRs were obtained from previously made plasmid constructs Plasmid expression vectors encoding MUC1 with 22 repeats (22TRMUC1) and two repeats (2TRMUC1) were

made in our laboratory (3) The cDNAs can be isolated from the plasmid vectors as HindIII cassettes (Fig 1) Plasmid expression vectors containing MUC1 cDNA with

42 TRs (42TRMUC1) and MUC1 cDNA without TRs (TR–MUC1), both BamHI

cas-settes, were obtained from Dr A Hollingsworth, University of Nebraska, Omaha 3.1.1 Cloning into pBlue Bac III Transfer Vector

An example we will use for cloning of the 3.2-kbp cDNA MUC1 with 22 TRs (22TRMUC1) and the 1.8-kbp cDNA MUC1 with 2 TRs (2TRMUC1) The resulting

pBlueBacIII-22TR-MUC1 recombinant transfer plasmid is used for inserting MUC1 cDNA into the genome of the wild-type Autographa californica Multiple Nuclear

Polyhedrosis Virus (wtAcMNPV), as described under Subheading 3.3.1.

1 Digest pBlueBacIII transfer vector with HindIII or BamHI and treat with calf intestine

phosphatase (CIP) to protect against self-ligation using standard methodology (see Note

1 and 2).

2 Prepare MUC1 cDNA cassette by HindIII digestion.

3 Purify fragments by electrophoresis in 0.7% agarose

4 Ligate the cDNA cassette into the pBlueBacIII vector using T4 DNA ligase at 16°C over-night

Table 1

MUC1 Specific Antibodies a

SM-3 IgG1 APDTRPb, underglycosylatedc

VU-4-H5 IgG1 PDTRPAP, underglycosylatedc

232A1 IgG Proteolytic cleavage site

aFor more details about antibodies see ISOBM TD-4 International Workshop

on Monoclonal Antibodies against MUC1, Tumor Biology, 1998, 19 (Suppl 1),

1–152

bSingle letter code for amino acids A, alanine; P, proline; T, threonine; D, glutamic acid; R, arginine

cTumor specific, recognizing underglycosylated but not fully glycosylated MUC1

5 Transform the ligated construct into E coli MAX Efficiency DH5α Competent Cells fol-lowing the protocol provided by the manufacturer

6 Select recombinants using Luria agar with 10 µg/mL of ampicillin (8).

7 Amplify ampicillin-resistant clones in 5-mL Luria broth/ampicillin (8) cultures Use 1.5–

3.0 mL of the culture to isolate plasmid DNA using Wizard Minipreps

8 Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert 3.1.2 Cloning into pFast Bac Transfer Vector

As an example, we will use cloning of 4.6-kbp cDNA with 42 TRs (42TRMUC1) Fragment of MUC1 cDNA coding for transmembrane and cytoplasmic domains was replaced with a sequence linking the outer membrane portion of MUC1 with glycosylphosphatidylinositol (GPI) anchor of human decay accelerating factor This

new construct (42TRMUC1-GPI) was made in our laboratory and remains as a BamHI cassette (Alter, M., unpublished data) The resulting pFastBac-42TRMUC1-GPI

recombinant transfer plasmid is used for inserting MUC1 cDNA into the genome of

the wtAcMNPV, as described under Subheading 3.3.2.

1 Linearize pFastBac transfer vector with BamHI digestion and protect it with CIP against

self-ligation using standard methodology

2 Cut out the 42TRMUC1-GPI cDNA cassette by BamHI digestion.

3 Purify a fragment of the correct size by electrophoresis in 0.7% agarose

4 Ligate the cDNA cassette into the vector using T4 DNA ligase at 16°C for overnight

5 Transform E coli MAX Efficiency DH5α Competent Cells with the ligated construct

6 Select recombinants using Luria agar with 10 µg/mL of ampicillin

7 Select ampicillin-resistant clones, and amplify and purify plasmid DNA using Wizard Minipreps

8 Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert

Fig 1 MUC1 cDNA expression plasmid The MUC1 cDNA is downstream from transla-tional start codon Constructs with 2 or 22 TRs that were made in our laboratory are contained

in the HindIII.

3.1.3 Cloning into the Episomal Transfer Vector pIE1-4

The vector pIEI-4 is used to provide stable expression of a cloned gene from the

baculovirus ie1 promoter Cells are cotransfected with pIE1-neo providing neomycin selection marker expressed from ie1 promoter As an example, we will use cloning of

1.4-kbp MUC1 cDNA that lacks TRs (TR–MUC1) The resulting pIE1-4TR– MUC1-GPI recombinant transfer plasmid is used for cotransfection of Sf-9 cells with

pIE-neo, as described under Subheading 3.5.

1 Linearize the pIE1-4 transfer vector with BamHI and protect it with CIP against

self-ligation using standard methodology (see Note 3).

2 Prepare TR–MUC1 cDNA cassette by BamHI digestion.

3 Purify the desired fragment by electrophoresis in 0.7% agarose

4 Ligate the cDNA cassette into the vector using T4 DNA ligase at 16°C for overnight

5 Transform E coli MAX Efficiency DH5α Competent Cells with the ligated construct

6 Select recombinants using Luria agar with 10 µg/mL of ampicillin Amplify ampicillin-resistant clones and purify plasmid DNA using Wizard Minipreps

7 Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert

3.2 Conditions for Culturing the Sf-9 Cells

Sf-9 insect cells are cultured in Hink’s TNM-FH Insect Medium supplemented with

5 or 10% FBS and penicillin/streptomycin/fungizone at the concentrations of 100 U/mL, 100 µg/mL, and 2.5 µg/mL, respectively Cells are grown as a monolayer at

27 °C For small scale growth, 75-cm2vented flasks are used (Costar, Cambridge, MA) Typically 5 × 105cells and 20 mL of medium are used to start the culture of this size For larger-scale growth, roller bottles are used Cultures are usually started at the cell density of 106cells/mL During the logarithmic phase of growth, cells typically double every 24 h Therefore, equal amounts of fresh medium are added each day to the roller

bottle for up to 300 mL total volume Figure 2 shows the kinetics of growth in a

typical roller bottle culture (see Note 4).

3.3 Production of Recombinant Virus by Cotransfection with Viral and Recombinant Transfer Vector DNAs

The wtAcMNPV viral DNA and the recombinant transfer vector DNA are shuttled into Sf-9 cells by cationic liposomes Within the cells, transfer vector DNA and viral DNAs recombine, incorporating the gene of interest into the viral genome Depending

on the transfer vectors different protocols can be used to make recombinant virus Two protocols are given next.

3.3.1 Using pBlue Bac III Vector

When using pBlueBacIII vectors, recombination leads to the replacement of the viral polyhedrin gene (phenotypically occ+) with part of the transfer vector containing

lacZ gene and gene of interest Therefore, the selection is based on the phenotypic observation—lack of occlusion bodies (occ-) and expression of β-galactosidase

(lacZ+).

1 Seed Sf-9 cells in a 6-well plate (106cells/well) prior to the cotransfection, and rock them gently side-to-side for 1 h at room temperature to evenly distribute and attach the cells

2 Remove nonattached cells and medium, gently wash the adherent monolayer once with serum-free medium, cover with 2 mL of serum free medium, and incubate for 30 min at room temperature

3 Prepare five independent transfection mixtures Mix 100, 200, 500, and 750 ng, or 1 µg of

the recombinant pBlueBacIII transfer plasmid, respectively, with 500 ng of linearized

AcMNPV DNA, 40 mL Insectin-Plus Liposomes, and 1 ml of Hink’s TNM-FH Insect Medium

4 Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min

5 Remove serum-free medium from the cells, cover cell monolayer with one of the transfec-tion mixtures, swirl to mix, and incubate for 4 h at room temperature with slow rocking

6 Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% FBS) to each well, wrap plates with clear plastic wrap, and incubate at 27°C for 48 h

7 Take 100 µL of the culture supernatant that contains viruses produced by the transfected cells

from each well and screen by plaque assay for the presence of double recombinants (occ -,

lacZ +) Transfer the remaining medium to sterile microcentrifuge tubes and store at 4°C 3.3.2 Using pFast Bac Transfer Vector

pFastBac transfer vector is a part of the Bac-To-Bac™ Baculovirus Expression

System developed by Gibco In the first step, competent MAX Efficiency DH10Bac

E coli cells are transformed with pFastBac donor plasmid with a gene of interest The competent DH10Bac E coli cells contain baculovirus shuttle vector (bacmid) and a

Fig 2 Growth of SF-9 cells in a typical roller bottle culture Infection was on d 4 and no new medium was added afterward On d 5 all cells were expressing β-galactosidase (see

Sub-heading 3.6.1 for details) Cells were usually harvested after 72 h.

helper plasmid Bacmid propagates in E coli and, besides viral DNA, contains several

other elements such as attachment sites for transposon Tn7 and an open reading frame

of lacZα peptide Recombination between pFastBac transfer vector and bacmid occurs

within bacterial cells by transposition, with the aid of helper plasmid Another feature

of this system is that insertion of the gene of interest into the viral genome causes

disruption of lacZ gene and E coli containing recombinant bacmid grow as white

colonies in the presence of Bluo-gal and IPTG This feature makes the system easy to use and eliminates posttransfection isolation of recombinant viruses.

1 Transform E coli MAX Efficiency DH10Bac Competent Cells that contain Bacmid DNA and helper plasmid After transformation with recombinant pFastBac-MUC1-42TR-GPI, the transposition occurs inside E coli cells disrupting LacZ gene within Bacmid DNA.

2 Select white growing colonies from Luria agar supplemented with kanamycin (50 µg/mL), tetracycline (10 µg/mL), gentamicin (7 µg/mL), BluoGal (100 µg/mL), and IPTG (40 µg/mL)

to make a larger amount of the recombinant Bacmid DNA

3 Amplify selected clones and purify Bacmid DNA using Wizard Minipreps

4 Seed 106Sf-9 cells/well in a 6-well plate immediately prior to transfection, and rock them gently side-to-side for 1 h at room temperature to evenly distribute and attach the cells Alternatively, seed 2.5 × 105/well and grow them until desired density, usually 1 to 2 d

5 Remove nonattached cells and medium, wash gently the cell monolayer once with serum free medium, cover with 2 mL of serum-free medium, and incubate for 30 min at room temperature

6 Prepare three independent transfection mixtures: 100 to 200 ng of Bacmid DNA, mixed with 20, 40, or 60 µL Insectin-Plus Liposomes, and 1 mL of Hink’s TNM-FH Insect Medium

7 Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min

8 Remove serum-free medium from the wells, cover cell monolayers with the transfection mixture, swirl to mix, and incubate for 4 h at room temperature with slow rocking

9 Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% of FBS) to each well, wrap with clear plastic wrap, and incubated at 27°C for 48 h

10 Transfer the culture supernatants that contain viruses produced by the transfected cells to sterile microtubes and store at 4°C

3.4 Isolation of Recombinant Baculovirus

The culture supernatants from cells cotransfected with the pBlueBacIII as a transfer

vector contain a mixture of recombinant and wild-type viruses To isolate recombi-nant viruses, a plaque assay is performed followed by an end-point dilution round of purification.

3.4.1 Plaque Assay

1 Seed Sf-9 cells in 6-cm dishes with 2 × 106cells/dish, and rock them gently side-to-side for 1 h at room temperature to evenly distribute and attach the cells

2 Grow cells at 27°C to approx 80% confluency Alternatively, seed more cells, and after 2

to 3 h during which the cells attach, the plates are ready for plaque assay

3 Make a serial 10-fold dilution of the medium harvested from transfected cells, in full Hink’s TNM-FH Insect Medium Dilutions should range from 10–1 to 10–5

4 Remove the medium from the wells and add 1 mL of the culture supernatant containing a mixture of wild-type and recombinant viruses (viral inoculum) to the side of the dish, and

tilt the dish slowly to cover evenly all cells It is important to do this gently in order not to disturb the attached cells Incubate at room temperature for 1 h

5 Melt the required volume of 3% SeaPlaque agarose, cool down to 45°C, and keep in a water bath Prewarm to 37°C an equal volume of Hink’s TNM-FH Insect Medium to which was added 120 µg/mL of X-gal

6 Remove the viral inoculum by tilting the plate and aspirating from the edge

7 Mix warm agar with medium and overlay dishes with 5 ml of this medium Leave leveled until agarose sets

8 Incubate at 27°C until blue plaques develop, usually 5–7 d

9 Using a sterile Pasteur pipet, pick isolated plaques with the recombinant virus (blue plaques without occlusion bodies), transfer to tubes containing 2 to 3 mL of Hink’s TNM-FH Insect Medium, and vortex for 30 s Allow viral particles to diffuse from agar for another hour at room temperature This could be used for screening and further purification

3.5 Production of an Episomal Recombinant Vector

for Stable Expression

3.5.1 Using pIE1-4 Vector

1 Seed Sf-9 cells in a 6-well plate (106cells/well) prior to the cotransfection, and rock them gently side-to-side for 1 h at room temperature to evenly distribute and attach the cells

2 Remove nonattached cells and medium, gently wash the adherent monolayer once with serum-free medium, cover with 2 mL of serum free medium, and incubate for 30 min at room temperature

3 Prepare five independent transfection mixtures Mix 3 µg of recombinant transfer

plas-mid pIE1-4-TR – -MUC1; 400 ng of pIE-neo plasmid DNA, and 20, 40, 60, 80, or 100 µL

of Insectin-Plus liposomes For mocktransfection, use 20 µL of Insectin-Plus liposomes

4 Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min

5 Remove serum-free medium from the wells, cover cell monolayer with the transfection mixture, swirl to mix, and incubate for 4 h at room temperature with slow rocking

6 Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% FBS) to each well, wrap the plates with clear plastic wrap, and incubate at 27°C for 48 h

7 Replace medium after 48 h with new medium containing 600 µg/mL of neomycin and grow cells for another 7 d

8 After 7 d cells can be tested for MUC1 expression by flow cytometry and Western blot

(see Notes 5 and 6).

3.6 Infection of Sf-9 Cells and MUC1 Production

3.6.1 Flask Cultures

1 Grow cells to approx 100% confluency

2 Aspirate all medium and cover the cell monolayer with the minimal volume of a viral

stock at 3 multiplicity of infection (see Note 7) This is usually 4 to 5 mL/75-cm2 flask

3 Rock the flask for 1 h at room temperature

4 Add 20 mL of serum-free medium and incubate at 27°C for a desired time To control

yield of infection with recombinant virus carrying lacZ gene, aliquot a small sample of

the culture (cells and supernatant) into a 1.5-mL microtube containing 1 µL of X-Gal at a concentration of 40 mg/mL, and incubate for 30 to 60 min at room temperature After that time, infected cells should exhibit blue color when observed microscopically owing to β-galactosidase expression, and the culture supernatant should turn blue If recombinant

virus does not carry and express lacZ gene, other signs of infection such as swollen nuclei

can be used to assess the efficiency of infection Typically more than 80% of cells are infected within the first day

3.6.2 Roller Bottle Cultures

1 Start a roller bottle culture with Sf-9 cells in 20 mL of in Hink’s TNM-FH Insect Medium,

at a density of 106cells/mL The cell number usually doubles once every 24 h Expand cells by adding every day an equal amount of fresh medium: 20 mL on d 2, 40 mL on d 3,

80 mL on d 4, and so forth, up to half the desired final volume of the culture (see Note 8).

2 Infect cells with the viral stock There are two procedures for infection of a roller bottle culture In the first, the culture is infected by adding the viral stock directly to the bottle and supplementing the culture with an equal amount of fresh medium 2 to 3 h after infec-tion The second method is to collect cells by centrifugation, resuspend in minimal vol-ume of medium, usually one-tenth of the original, add the viral stock, and rock for 1 h at room temperature Transfer infected cells back to the bottle, and add fresh medium in the amount equal to that prior to infection We found both methods worked equally well The only differ-ence is that in the first method, in order to accomplish complete infection of all insect cells within 24 h, cultures should be infected at 5 m.o.i or higher In the second method, although more laborious, less viral stock is used and infection at 3 m.o.i is sufficient

3.7 Starving Sf-9 Cells in Culture

to Obtain Underglycosylayted Forms of MUC1

3.7.1 Flask Cultures

1 Grow cells to 100% confluency, and prolong the time of culture for another day or two without changing the medium in order to deplete most of the nutrients At this time, the number of dead cells slightly increases

2 Aspirate all medium, clear by centrifugation and filtration through a 0.22-µm filter, and save

3 Cover the cell monolayer with a minimal volume of the viral stock, usually 4 to 5

mL/75-cm2flask

4 Rock the flask for 1 h at room temperature

5 Add the saved nutrients-depleted medium and incubate the culture at 27°C for a desired

time To control efficiency of infection with recombinant virus carrying lacZ gene,

fol-low the procedure described under Subheading 3.6.2.

3.7.2 Roller Bottle Cultures

1 Start and expand a roller bottle culture as described under Subheading 3.6.

2 Starve cells by growing at high density for 48 h without supplementing with fresh medium

3 Infect cells with a viral stock as described under Subheading 3.6 In starved cultures, if

cells are harvested for infection, nutrient-depleted medium should be collected, cleared

by centrifugation and filtration through a 0.22-µm filter, and added back to the bottle

3.8 Analysis of Recombinant MUC1 Produced by Sf-9 Cells

The quickest method to test the expression of MUC1 in insect cells is by immunostaining with specific MAbs There is a large number of well characterized MAbs against different peptide and sugar epitopes on MUC1 Western blot is the method of choice for testing total expression, and flow cytometry is used for testing cell surface expression The pattern of reactivity of baculovirus expressed MUC1 with

various anti-MUC1 MAbs does not reflect the degree of glycosylation of the recombi-nant product Although we do not provide a detailed protocol for Western blot and flow cytometry in this chapter, some technical details could be found in the figure

legends (see Note 9).

MUC1 is the only transmembrane mucin known to date It is transported to the apical surface of the ductal epithelial cells and anchored in the cell membrane via its transmembrane domain MUC1 is removed from the cell surface by proteolytic cleav-age in the membrane proximal domain, or, to lesser extent, it is internalized and degraded in the phagolysosomes In insect cells, MUC1 also undergoes complex pro-cess of posttranslational modification as in mammalian cells It is also transported to

the surface of Sf-9 cells (6).

A single, large band with an apparent molecular weight above the 221 kDa, repre-sents the only form of MUC1 expressed by insect cells when they are grown in fully supported medium containing 10% FBS However, when cells are starved for at least two days prior to infection and then grown in nutrient-depleated medium, the majority

Fig 3 Different forms of MUC1 expressed in Sf-9 cells using recombinant baculovirus

Form designated as (A) is a protein precursor which undergoes a proteolytic modifiction yeilding a 20 kDa smaller protein (B) Form (C) has not been characterized yet, and form (D) is

MUC1 released from the cell surface