J. Cell Sci. 12, 585-602 (1973) 585Printed in Great Britain

INTESTINAL GOBLET CELL MUCUS: ISOLATION

AND IDENTIFICATION BY IMMUNO-

FLUORESCENCE OF A GOBLET

CELL GLYCOPROTEIN

J. FORSTNER, N. TAICHMAN, V. KALNINS AND G. FORSTNER*Gastrointestinal Unit, Toronto Western Hospital, and Departments of Medicine,Anatomy and Experimental Pathology, University of Toronto, Canada

SUMMARY

A high-molecular-weight glycoprotein (HMG), representing the majority of the solubleglycoprotein hexosamine and hexose in the intestine, was isolated by Sepharose 4B chroma-tography from high-speed supernatants of rat small intestinal homogenate. Fluorescein-labelled globulin, from rabbit antiserum produced against HMG, specifically stained supra-nuclear mucus vesicles of goblet cells in small intestine and colon as well as gastric pit cells inthe body of the stomach and mucus-producing cells in the sublingual salivary gland. A singleprecipitin line was found when HMG was tested against its antibody by agar immunodiffusionand immunoelectrophoresis. No cross-reactivity was observed between antibody and rat serumor extracts from microvillus membrane, human colon and pig intestine. Precipitin lines whichfused with the HMG precipitin arc in apparent identity were observed with antigens inintestinal lumen washings, and in small-molecular-weight fractions from intestinal cell sap.When studied by cellulose acetate electrophoresis, cetyl trimethylammonium bromide precipi-tation and precursor labelling with [i-14C]glucosamine, HMG behaved as a single homo-geneous glycoprotein free of detectable protein contamination. These results imply that HMGis a major component of goblet-cell mucus in the small intestine, and suggest that it is similarto mucin produced throughout the gastrointestinal tract. HMG is the first glycoprotein,isolated without the aid of proteolytic agents, which has been specifically identified as a productof the goblet cell.

INTRODUCTION

Glycoproteins of the glycocalyx and mucus are thought to provide a primarybarrier to solutes, enzymes, bacteria and chemicals within the lumen of the intestinaltract (Hollander, 1954; Ito, 1965; Spiro, 1963; Trier, 1968). This role has not beenadequately examined however, for want of isolated, well-identified native glycopro-teins suitable for the examination of physical and chemical behaviour. In previousstudies (Forstner, 1968, 1970) an attempt was made to separate glycoproteins presentwithin rat intestinal mucosal homogenates into separate classes on the basis of theirsubcellular localization, and incorporation of the precursor sugar [i-14C]glucosamine.Glycoproteins specifically attached to the microvillus membrane were identified, andsome of these were shown to be surface disaccharidases (Forstner, 1971). On a

• Address for reprint requests: Dr J. Forstner, Clinical Science Division, Medical SciencesBuilding, Room 6356, University of Toronto, Toronto, Ontario, Canada.

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586 J. Forstner, N. Taichman, V. Kalnins and G. Forstner

quantitative basis, however, the major glycoprotein class was a soluble, poorly labelledfraction isolated from the post-microsomal supernatant by Sepharose 4B chroma-tography (Forstner, 1970). This fraction accounted for 69% of the cell sap glyco-protein hexosamine. Since it was eluted from Sepharose 4B near the void volume itappeared to represent a class of glycoproteins of very high molecular weight.

In the present study the high-molecular-weight peak has been characterized furtherand shown to represent a glycoprotein localized in the goblet cell and the extracellularmucus coat. Antigenically similar material was present in intestinal washings, colon,stomach and salivary gland, as well as in smaller-molecular-weight fractions of theintestinal cell sap. The mild techniques used in its preparation suggest that thisglycoprotein will be of use in the study of the physical and functional characteristicsof native intestinal mucus.

MATERIALS AND METHODS

Preparation of high-molecular-weight glycoprotein {HMG)

HMG was prepared initially from the post-microsomal supernatant of rat small intestinalmucosa as previously described (Forstner, 1970) using a small Sepharose 4B column (0.9 x48 cm). The eluate from 11-14 ml was combined. In later experiments, batch preparation wasachieved by a modified procedure. The intestinal scrapings from 3 rats were homogenized in aWaring blender for 20 s in 5-0 DIM EDTA adjusted to pH 7-4 with NaOH (100 ml for each gwet weight of tissue). The homogenate was centrifuged at 36000 g for 30 min, the supernatantdialysed against distilled water at 4 °C for 24 h, lyophilized and suspended in 10 ml of distilledwater. The suspension was recentrifuged at 36000 g for 30 min. The final supernatant (derivedfrom an original wet weight of 10 g of mucosa) was then applied to a column of Sepharose 4B(85 x 2-5 cm) and eluted with 10 mM K,HPO4-KH,PO4 buffer, pH 7-0 and a hydrostaticpressure of 9-8 x io* N m~* (10 cm H,O). The eluate from 125-185 ml was pooled, dialysedagainst distilled water for 24 h at 4 °C and lyophilized. The HMG product was identical to thatinitially prepared from the post-microsomal supernatant by all the criteria discussed in thispaper.

Preparation of antiserum to HMG

HMG antigen was prepared from 10 separate eluates from a small Sepharose 4B column(0-9 x 48 cm) as above, and the combined pool dialysed against distilled water for 24 h, lyophil-ized and suspended in phosphate-buffered saline to give a final concentration of 10 mg/ml.

Antiserum to HMG was obtained from 5 healthy adult female New Zealand rabbits by amodification of the method described by Kopp, Trier, MacKenzie & Donaldson, Jr (1968).Approximately 30 mg of HMG was suspended in 3 ml of phosphate-buffered NaCl, pH 70,mixed vigorously with an equal volume of Freund's adjuvant (Difco Labs) by repeated passagethrough a 20-gauge needle. This freshly prepared suspension was injected subcutaneously in1-ml aliquots into the back and toe pads of each rabbit. A similar injection was given 3 weekslater. Two weeks after the last injection serum was obtained from the rabbits by cardiacpuncture.

Preparation of fluorescein-labelled antibody

Following the procedure of Nairn, Fothergill, McEntegart & Porteous (1962), serumglobulins were precipitated by 40 % saturation with ammonium sulphate, dialysed againstphosphate-buffered NaCl, and adjusted to a concentration of 2 % (w/v). Conjugation of 4 mlof 2 % globulin was achieved by mixing it with 0-0125 mg fluorescein isothiocyanate (BaltimoreBiological Laboratories, Baltimore, Md.) per mg of globulin for 30 min at pH 953. The

Goblet cell mucus 587

resulting solution was then passed through Sephadex G-50 in 0125 M NaCl in 0-0175 Mphosphate buffer pH 6-3 to remove free fluorescein. The first coloured peak was concentratedby pressure dialysis to 4-0 ml, and applied to a column of DEAE-cellulose (1-5 x 15 cm) whichhad previously been washed with 00175 M phosphate buffer pH 6-3. The fiuorescein-labelledgammaglobulin was eluted with 0-125 M NaCl in 00175 M phosphate buffer pH 6-3 as outlinedby Riggs, Loh & Eveland (i960), and concentrated by pressure dialysis to 10 mg/ml. Themolar fluorescein: protein ratio was about 2-0.

Immunofluorescent studies

Tissue segments approximately 05 cm in the longest diameter were fixed in 10% neutralbuffered formalin for 4-0 h at room temperature and left in 30% sucrose in water overnight at4 °C (Kopp et al. 1968). The segments were embedded in O.C.T. compound (Ames Labora-tories, Elkhart, Ind., U.S.A.) and sectioned at — 20 °C. Sections 4-6 /im thick were fan-driedon microscope slides, incubated with fiuorescein-labelled globulin for 30 min at room tem-perature, washed thoroughly with 2 changes of phosphate-buffered saline for 20 min, coveredwith buffered glycerol and sealed with a coverslip. Specimens were examined with darkfieldillumination through a Zeiss fluorescence microscope.

Immunodiffusion studies

To test the reactivity of immune rabbit globulin against rat HMG and other isolated glyco-proteins or antigens, double diffusion was performed by the method of Ouchterlony (1968), in0-9 % agar in 0-075 M veronal acetate buffer pH 8-6 on glass microscope slides. The reactionswere allowed to develop at room temperature for 24-48 h, the agar on the slides was thenwashed with isotonic saline and tap water, and subsequently dried and stained with amidoblack.

In addition to HMG other materials tested in immunodiffusion studies were obtained asfollows, (a) Rat microvillus membranes were prepared as described previously (Forstner,Sabesin & Isselbacher, 1968), and then solubilized in 0-5 % sodium deoxycholate (DOC).(6) Human colon material was prepared by homogenization of mucosa in 4 ml of 0-5 % DOCwith Potter-ElvejhemR glass homogenizers. After standing in DOC for 2 h at room temperatureboth (a) and (b) were centrifuged at 28000 g for 30 min to remove paniculate material. Thesupernatants were stored at — 20 °C until use. (c) Samples of Vitamin A-dependent goblet-cellfucose-containing glycopeptide (DeLuca, Schumacher & Nelson, 1971) and antiserum pre-pared in chickens were kindly supplied by Dr L. DeLuca. (d) Jejunal lumen material wasobtained by washing isolated gut segments with isotonic saline. Washings were centrifuged at30000 g for 20 min, dialysed against distilled water for 24 h, concentrated by lyophilization,and frozen until use.

Immunoelectrophoresis

Immunoelectrophoresis was performed in 0-9 % agar in 0-075 M veronal acetate buffer,pH 8-6, on glass microscope slides in a Gelman electrophoresis chamber for 60 min at 10 mAper frame; 25 ji\ of immunoglobulin were then added to the centre trough and the slidesstained with amido black as described above.

Cellulose acetate electrophoresis

Cellulose acetate electrophoresis was performed in 0075 M veronal acetate buffer pH 86 onSepraphore III for 40 min in a Beckman electrophoresis chamber at 250 V. Volume of sampleswas 1 /A. Strips were stained with the Ponceau-S reagent for protein, and periodic acid/Schiff(PAS) reagent for glycoprotein as described by Gelman (Electrophoresis Manual).

Precipitation of HMG with cetyltrimethylammonium bromide (CTAB)

One per cent CTAB in distilled water was added to 10 ml distilled water containing 2 6 mgH M G protein to produce a final volume of 120 ml and a CTAB concentration of o-i %. The

38-2

588 J. Forstner, N. Taichman, V. Kalnins and G. Forstner

A

-1 0 2

0-1

125 250 375 500

Fig. 1. Sepharose 4B column chromatography of rat intestinal cell sap. Column size,and conditions of operation are as described in Methods and Results. Hexose (O—O) ,counts per min (x — x ), protein ( • — # ) , Maltase (A), sucrase (B) and alkalinephosphatase (C) activities were determined as described in Methods. The arrowindicates the void volume as determined by Blue Dextran 2000. Abcissa shows eluatevolume in ml.

mixture was left standing at room temperature overnight, the precipitate collected by centrifu-gation at 1000 g for 20 min, and washed twice with 10 ml of 0 1 % CTAB. The combinedsupernatant and washing solutions were dialysed against 0-15 M NaCl and then against wateralone, lyophilized and resuspended in 1 ml of water. The pellet was solubilized by the additionof 2 ml of 0-15 M NaCl, dialysed against 015 M NaCl for 24 h and then against water for 24 h.After lyophilization the 'soluble' pellet was resuspended in 1 ml of watei.

Determination of critical salt concentration of the HMG-CTAB complexThe method of Scott (i960) was used. H M G (approximately 3-6 mg hexose) was completely

dissolved in 14-0 ml of 0-16 M NaCl containing 0-4% CTAB, and then diluted stepwise withdistilled water. After thorough mixing the precipitate was collected by centrifugation at 1000 gfor 30 min. Hexose was assayed after solubilization of the precipitate in 0-16 M NaCl.

AssaysHexose was determined with anthrone (Spiro, 1966). Glucosamine and maltase, sucrase and

alkaline phosphatase activities were determined as described previously (Forstner, 1971).

RESULTS

Chromatographic separation of HMG on Sepharose 4B

The supernatant from a rat intestinal homogenate labelled 2 h prior to sacrifice withintraperitoneally injected [i-14C]glucosamine was obtained and applied to a Sepharose4B column (85 x 2-5 cm) as described in Methods. The resulting elution profile is

Goblet cell mucus 589

shown in Fig. 1. The major hexose peak appeared at the void volume and was associ-ated with single radioactivity and protein peaks. The centre of this peak was appro-priately combined as indicated in Fig. 1, concentrated by lyophilization, and stored at— 20 °C for further use. This fraction is subsequently referred to as high-molecular-weight glycoprotein (HMG). Aliquots serially removed from across the HMG peakwere also assayed for hexosamine and hexosamine specific radioactivity. As suggestedfrom the coincident hexose and radioactivity peaks in Fig. 1, hexosamine specificradioactivity was relatively constant throughout the peak, varying from 1-2 to 1-4 cpmper fig. Two additional fractions, F2, consisting of the tail of the HMG peak, and F3,consisting of the major radioactivity peak, were also combined as shown in Fig. 1 andsimilarly concentrated. The molecular-weight range of proteins within these fractionswas estimated roughly from comparison with the elution profiles for maltase, sucraseand alkaline phosphatase, which have estimated molecular weights of 400000, 200000and 70000 respectively (Forstner, 1971). F3 ranges in molecular weight from slightlyless than 70000 to about 400000 while the lower limit for F2 is approximately 400000.

Immunofluorescent localization of HMG

When rat intestinal sections were incubated with fluorescein-labelled immunerabbit globulins, fluorescent staining was localized to the supranuclear, vesicularregion of goblet cells in the villi (Figs. 3, 6) and the crypts (Fig. 5) of the jejunum andileum. Mucus secretions of goblet cells also appeared to be stained (Fig. 3). Fluores-cence could be detected in cells at the base of the crypts (Fig. 5), as well as in themore distended goblet cells located further away from the base of the crypts and inthe villi (Figs. 3, 6). A conspicuous fluorescent column or layer was always presentwithin the crypt lumen (Fig. 5), even if the tissue was washed with 0-15 M NaClprior to incubation with antibody. In the case of villi, however, a continuous layer offluorescent luminal material was only seen in tissues which had not been everted, orextensively washed with NaCl (Fig. 6). The underlying brush border exhibited nospecific fluorescence in areas which were denuded of mucus (Fig. 3). As a control,fluorescein-conjugated normal rat serum globulin did not stain goblet cells in thecrypts or in the villi (Fig. 5) of the small intestine; neither did it stain mucus adherentto the brush border. In Fig. 11, several crypts from the small intestine in cross-sectionare strongly stained by labelled immune globulin. Preincubation of the immuneglobulin with HMG (200 fig per ml) before application to an adjacent tissue sectionresulted in marked diminution of staining (Fig. 12). Preincubation of the tissue slicewith unlabelled immune globulin also reduced staining.

In the colon, the fluorescein-labelled antibody also stained goblet cells and themucus in the crypt space (Fig. 8). Non-immune fluorescein-labelled globulins didnot stain either of these (Fig. 6). Specific fluorescence was also detected in cells lininggastric pits in the body of the stomach (Fig. 9) and the mucus cells of the sublingualsalivary gland (Fig. 10). Thus the same antigenic determinant(s) appeared to be sharedby mucus produced throughout the rat gastrointestinal tract.

590 J. Forstner, N. Taichman, V. Kalnins and G. Forstner

Specificity of the antibody for HMG

Immunodijfusion and immunoelectrophoresis. The antiserum, when reacted againstthe post-microsomal supernatant (cell sap) and HMG, gave single precipitin lineswhich fused in a reaction of identity (Fig. 13). No reaction occurred with control ratserum (RS), human colonic homogenate (HC), DOC-solubilized microvillus mem-branes (MM) of rat intestine, or goblet cell' fuc-glycopeptide' of DeLucaei al. (1971).In other experiments (not shown) no precipitin line was observed with soluble cell-sap proteins from porcine intestine, while antisera to the 'fuc-glycopeptide' failedto react with HMG.

As shown in Fig. 14, a reaction of identity was observed between HMG, fractions2 and 3 from the Sepharose 4B column, the original cell sap (CS), and the intestinallumen contents (LUM). A second faint precipitin line, closer to the central well, wasseen with the LUM and F3 material although it is not well shown in Fig. 14. Thisprecipitin line fused with the single line of CS and HMG, suggesting that a serologic-ally related but faster-diffusing antigen was being detected. This precipitin line dis-appeared much more rapidly than the major precipitin line with dilution of eitherantigen or antibody.

HMG, CS, F2 and F3 each elicited a single precipitin arc on immunoelectrophoresis.They all had similar electrophoretic mobility (Fig. 15). The longest arc was seen withF3, in keeping with the smaller molecular weight and greater diffusibility of theglycoproteins in this fraction.

Cellulose acetate electrophoresis. HMG gave a broad, homogeneous PAS-positiveband which possessed slight anodic mobility (Fig. 16). In contrast, both F2, F3 andCS contained a PAS-positive band with greater anodic mobility than HMG. At alltimes small amounts of PAS-positive material could be detected at the origin as well.When amounts applied to the origin were increased to make protein detection possiblein HMG, the majority of the protein and periodic acid-Schiff positive material becamefixed at the origin (Fig. 16). Under these circumstances protein and periodic acid-Schiff staining was at least confined to the same area so that the presence of proteincontaminants of greater mobility can be excluded. Fig. 17 also demonstrates that thecell sap contains anodic and cathodic proteins which migrate well beyond the glyco-protein bands.

Precipitation vrith cetyltrimethylammonium bromide (CTAB). As an alternative testof the homogeneity and purity of HMG related directly to the anionic polyelectrolyteproperties expected of an epithelial mucin, HMG was precipitated with a long chainquaternary ammonium base cetyltrimethylammonium bromide (CTAB). Table 1illustrates that virtually all of the protein, hexose and hexosamine in HMG was pre-cipitated by CTAB. The precipitate was completely soluble in 0-15 M NaCl. Recoveryafter solubilization of the precipitate in 0-15 M NaCl, dialysis against distilled H20and concentration by lyophilization was incomplete but amounted to 80% of theprotein and 85% of the hexosamine. These results make it extremely unlikely that theHMG preparation contains significant amounts of non-glycoprotein protein. Thesolubility of the CTAB-glycoprotein complex at low NaCl concentration also indicates

Goblet cell mucus

Table 1. Precipitation of HMG by o-1 % cetyltrimethylammonium bromide

Soluble beforeprecipitation,

mg

Soluble afterprecipitation,

mg

Amountprecipitated,

0//o

ProteinHexoseHexo8amine

2-642-843392

0-0810-1140-046

96-896-0987

Conditions of incubation of HMG with CTAB, and assays were as described in Methods.The amount precipitated is the difference between values obtained for HMG before additionof CTAB, and the amount present in the combined supematants after precipitation.

10

M

OX

0-5

005 01 0-15

NaCI cone, M0-2

Fig. 2. Precipitation of HMG—CTAB complex at critical salt concentration bystepwise dilution with water in excess CTAB. Method and results are as described intext. Hexose (#—%) determined in the precipitate with anthrone reagent.

that the entire preparation has the weakly anionic polyelectrolyte behaviour charac-teristic of epithelial mucins (Scott, i960; Antonopoulos et al. 1961).

The CTAB-precipitated and subsequently solubilized HMG preparation wasindistinguishable from untreated HMG on cellulose acetate electrophoresis (notshown). On immunodiffusion the treated HMG preparation appeared to diffusepoorly out of the antigen well so that only a poor precipitin line at the well marginwas obtained. However, incubation of specific antiglobulin with the treated HMGresulted in complete absorption of antibody against unprecipitated HMG. Thus theantigenic activity of HMG was retained through precipitation with CTAB.

Fig. 2 illustrates the precipitation curve obtained when a separately preparedHMG-CTAB complex was solubilized with 0-16 M NaCI, and serially diluted withdistilled water, in the presence of excess CTAB (Scott, i960). Maximum precipitation

592 J. Forstner, N. Taichman, V. Kalnins and G. Forstner

occurred sharply at o-i M NaCl. The curve was also symmetrical indicating a singlecritical salt concentration for most of the CTAB complexes.

DISCUSSION

A major problem in using immunofluorescence to study epithelial mucins is therequirement for a homogeneous single antigen for use in eliciting a single antibody(Kent, 1968). HMG appears to be homogeneous by at least 5 criteria. It forms asingle, somewhat broad band on cellulose acetate electrophoresis. It is free of a majorcontaminating protein, as judged by electrophoresis, and by its total precipitation withcetyltrimethylarnmonium bromide. HMG-CTAB complexes have a common criticalsalt concentration in NaCl, suggesting a minimum of charge heterogeneity. Injectionof the glycoprotein precursor [i-14C]glucosamine in vivo results in radioactive labellingof HMG hexosamine which is virtually constant throughout its chromatographic peak.HMG gave a single precipitin line in immunodiffusion and immunoelectrophoresis testswith an antibody from rabbit serum which was obtained by immunization with HMG.

In spite of this apparent homogeneity a second minor precipitin line was obtainedwhen the HMG-specific antibody was tested by immunodiffusion against a concen-trated small-molecular-weight antigen from fraction F3 from the Sepharose 4Bcolumn. HMG therefore may have contained 2 types of antigen. In quantitative termsthe second reactive antigen in fraction 3 is unimportant since the entire fractionrepresents less than 10% of the cell sap glycoprotein (as hexose). The possibility thatit may represent a subunit or degradation product of HMG is currently underinvestigation.

By immunofluorescence HMG is specifically located in goblet cells in the intestine,and in the adherent mucus layer in crypts and on the surface of intestinal villi. It istherefore probably a major glycoprotein, if not the only glycoprotein in the secretionof the goblet cell. The fluorescein-labelled antibody also specifically stained mucusand mucus-producing cells in the colon, stomach and salivary gland, indicating thatHMG has antigenic determinants which are shared by mucus throughout the gastro-intestinal tract. A similar type of cross-reactivity between epithelial mucins fromdifferent organs has been observed by L. DeLuca using the immunofluorescencetechnique (personal communication), and may imply that an identical mucin issecreted by many different epithelial tissues. Exceptions have also been noted however.For example, Kent (1961) has reported the production of an antibody to bovinesalivary mucin which reacted in immunofluorescence tests with bovine submaxillarygland but not with bovine stomach, colon or gallbladder.

HMG antibody did not react with an extract from human colon (Fig. 13) or with anextract from pig intestine. These results do not necessarily imply species specificitysince the preparative technique used in obtaining the antigen may be critical. It isnoteworthy, for example, that no cross-reactivity was seen with the rat fucose-glycopeptide isolated by DeLuca, Schumacher & Wolf (1970). This fucose-glycopep-tide has also been localized in the goblet cells of the rat intestine by immunofluores-cence, and its behaviour on Sepharose 4 B suggests that it is a large-molecular-weight

Goblet cell mucus 593

glycoprotein similar to HMG. The explanation for the lack of immunological cross-reactivity with HMG may lie in the fact that the fucose-glycopeptide was exposed toextensive proteolysis prior to its isolation, and may have lost the antigenic determin-ants present on HMG.

Proteolysis, sulphitolysis, phenol extraction, ethanol, urea and other agents arefrequently used in isolating soluble epithelial mucins (Pamer, Jerzy Glass & Horowitz,1968; Kim & Horowitz, 1971; Inou6 & Yosizawa, 1966). The products obtained havevaried and there has been little assurance that they closely resemble the parentmaterial. The mild techniques used in the isolation of HMG suggest that it is likelyto resemble native goblet cell mucin closely in its physiochemical behaviour. Similarclaims have been made for the soluble mucins isolated by Snary & Allen (1971),Katzman & Eylar (1966) and Kim & Horowitz (1971). Of these, HMG is the onlymucin which has actually been localized in a mucus secreting cell.

As well, it is worth noting that sulphitolysis, which was used as a solubilizingtechnique by Kim & Horowitz (1971), may have introduced an artifactual disruptionof an otherwise functionally homogeneous glycoprotein by breaking interchain disul-phide linkages. Katzman & Eylar (1966) separated porcine submaxillary mucin into2 fractions by anion exchange chromatography, but this method of separating glyco-proteins has been questioned (Inoue & Yasikawa, 1966), on the grounds that subunitsof the mucin may be separated as a result of microheterogeneity. For the present thesomewhat arbitrary use of preparative procedures relying on molecular weightdifferences seems preferable in isolating native mucins on the assumption that thelargest molecules are disrupted least and are therefore most likely to represent theoriginal material. In this context it is clear that antigenically similar glycoproteins ofmuch smaller molecular weight than HMG exist in the intestinal homogenates.Whether these represent forms present in the living cell or fragments removed duringpreparation cannot be answered.

In our previous studies the HMG fraction of the cell sap was shown to be poorlylabelled by [i-14C]glucosamine in comparison with surface membrane and secretedglycoproteins. As a result it was suggested that HMG fraction had a very slow turnoverrate. Initially we rejected the possibility that HMG represented a storage mucin in thegoblet cell since autoradiographic studies had shown that labelled mucins in the gobletcell were discharged into the intestinal lumen almost as quickly as labelled glycopro-teins appeared on the plasma membrane (Neutra & Leblond, 1966; Ito, 1969). Sincethe present studies have clearly shown that HMG is present in the goblet cell, thisconclusion must be re-evaluated. If HMG is a highly polymerized storage mucin, it istempting to postulate that the antigenically related small-molecular-weight glycopro-teins in the F3 fraction might constitute rapidly labelled and secreted goblet cellglycoproteins identified by autoradiography. The relative labelling patterns of bothlarge- and small-molecular-weight glycoproteins is currently under investigation inorder to clarify this point.

594 J- Forstner, N. Taichman, V. Kalnins and G. Forstner

We gratefully acknowledge the assistance of Miss I. Jabbal, Mrs A. Madapallimattam andMrs L. Subrahmanyan. This work was supported by grant no. MA 2340 of the MedicalResearch Council of Canada, and the Canadian Cystic Fibrosis Foundation. Dr J. Forstnerwas a Canadian Cystic Fibrosis Foundation Fellow.

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Washington, D.C.: American Physiological Society.(Received 28 June 1972)

596 J. Forstner, N. Taichman, V. Kalnins and G. Forstner

Fig. 3. Adjacent jejunal villi, x 400, stained with fluorescein-labelled immunoglobulinagainst HMG. Specific fluorescence i9 localized in goblet cells, and surface clumps ofmucus. The specific fluorescence of mucus clumps can be distinguished from auto-fluorescence of areas of the brush border surface which can also be seen in Fig. 4.Some non-specific fluorescence can be seen in lamina propria.Fig. 4. Jejunal villus, x 400, stained with fluorescein-labelled non-immune globulin.Non-specific fluorescence can be seen in areas of brush border surface and also in thelamina propria. Goblet cells are not stained.Fig. 5. Jejunal crypt, x 400, stained with fluorescein-labelled immunoglobulin (anti-HMG). Cells at the base of the crypt (cb) exhibit apical, loosely clumped stainingwhile full goblet cells appear in the fourth or fifth layer of cells from the bottom ofthe crypt. The lumen of the crypt is filled with stained secretion from goblet cells.Fig. 6. Adjacent jejunal viUi, x 400, from uneverted minimally washed intestinestained with fluorescent immune globulin (anti-HMG). Note the continuous layer offluorescently stained material in space between villi, and the stained goblet cells, someof which (arrows) are secreting stained material into the lumen.

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598 J- Forstner, N. Taichman, V. Kalnins and G. Forstner

Fig. 7. Rat colon, x 400, stained with non-immune fluorescein-labelled globulin.Note absence of fluorescence in goblet cells.Fig. 8. Rat colon stained with fluorescein-labelled immune globulin (anti-HMG),x 400. Goblet cells and surface mucus are heavily stained.Fig. 9. Rat body of stomach, stained with fluorescein-labelled immune globulin (anti-HMG), x 200. Cells bordering mid and deeper regions of pits are stained as well asan occasional clump of secreted mucus on the surface.Fig. 10. Rat sublingual gland, stained with fluorescein-labelled immune globulin(anti-HMG), x 200. Staining is confined to cells in mucus-producing glands.Fig. 11. Ileal crypt in cross-section stained with fluorescent immune globulin (anti-HMG), x 400. Goblet cells are strongly stained.Fig. 12. Ileal crypt in cross-9ection stained with fluorescein-labelled globulin (anti-HMG) preincubated with HMG, x 400. Note markedly decreased fluorescence ofgoblet cells.

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6oo J. Forstner, N. Taichman, V. Kalnins and G. Forstner

Fig. 13. Immunodiffusion in agar of anti-HMG (1:10 dilution) in centre well andvarious antigens, showing single precipitin line against CS (cell sap) and HMG withfusion. MM, microvillus membranes; HC, human colon; D, ' fuc-peptide' of DeLuca;RS, rat serum.Fig. 14. Immunodiffusion in agar of anti-HMG in centre well (1:10 dilution) andvarious antigens, showing fusion of precipitin lines for CS, HMG, Fs, F3 and luminalmaterial (LUM). The second precipitin line formed with Fs and lumen material ispoorly seen.Fig. 15. Electrophoresis of HMG, CS, F, and F,. Precipitin lines formed againstanti-HMG (1:5 dilution).Fig. 16. Cellulose acetate electrophoresis of CS, HMG, F2 and F, stained with PASto demonstrate glycoprotein. Arrow marks the point of application.Fig. 17. Cellulose acetate electrophoresis of CS, HMG with super-loading to demon-strate protein as well as carbohydrate. One specimen stained with Ponceau-S forprotein, the other with PAS. Arrow marks the point of application.

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