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Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells.

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THE ANATOMICAL RECORD 230:307-318 (1991)
Differential Distribution of Salivary Agglutinin and
Amylase in the Golgi Apparatus and Secretory
Granules of Human Salivary Gland Acinar Cells
KUNIO TAKANO, MEREDITH BOGERT, DANIEL MALAMUD, EDWARD LALLY,
AND ARTHUR R. HAND
Department of Oral Histology, Nagasaki University School of Dentistry, Nagasaki 852,
Japan (K.T.);Center for Oral Health Research, University of Pennsylvania School of
Dental Medicine, Philadelphia, Pennsylvania 19104 (M.B., D.M., E.L.); Clinical
Investigations and Patient Care Branch, National Institute of Dental Research, NIH,
Bethesda, Maryland 20892 (A.R.H.)
ABSTRACT
The secretory granules of salivary glands often display complex
internal substructures, yet little is known of the molecular organization of their
contents or the mechanisms involved in packaging of the secretory proteins. We
used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and a-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells.
With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin,
reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions
of low and medium electron density, but not in the dense cores. Reactivity seen on
the apical and basolateral membranes of acinar and duct cells was attributed to a
shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody
to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in
the dense cores and in areas of medium density, but not in the regions of low
density. These results indicate that these two proteins are distributed differently
within the secretory granules, and suggest that they follow separate pathways
between the Golgi apparatus and forming secretory granules. Small vesicles and
tubular structures that labeled only with the antibodies to the agglutinin were
observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in
transport of the putative membrane glycoprotein to the cell membrane.
Abundant evidence has recently been obtained demonstrating the differential regulation of protein transport and secretion in eukaryotic cells. Key regulatory
points in this process are a t the level of ER-Golgi apparatus transfer (Rose and Doms, 1988), transport and
post-translational modifications in the Golgi apparatus
(Kornfeld and Kornfeld, 1985; Farquhar, 1985; Pfeffer
and Rothman, 1987; Rothman and Orci, 1990), and
sorting and packaging into vesicles or granules destined for constitutive or regulated discharge (Kelly,
1985; Burgess and Kelly, 1987). In spite of considerable
recent attention (Chung et al., 1989; Stoller and
Shields, 1989), relatively little is known a s yet of the
mechanisms involved in the sorting and packaging of
proteins into the characteristic granules found in regulated secretory cells (Burgess and Kelly, 1987).
The acinar cells of salivary glands have served a s
useful models for studying various aspects of the secretory process. Unlike other exocrine glands, salivary acinar cells show a remarkable diversity of morphological appearances among glands and among species
(Young and van Lennep, 1978). In particular, the struc0 1991 WILEY-LISS, INC
ture of secretory granules is highly variable. They
range in appearance from electron-lucent to highly
electron-dense, and their content may be homogeneous
or display regions of variable density, often arranged in
complex patterns (e.g., Tandler and MacCallum, 1972).
The granules of acinar cells of human parotid and submandibular glands typically contain dense cores, crescent-shaped structures, andlor strands of dense material in a skein-like pattern (Tandler and Erlandson,
1972; Riva and Riva-Testa, 1973; Riva et al., 1974).
Received April 16, 1990; accepted December 5, 1990.
Dr. Arthur R. Hand’s present address is Department of Pediatric
Dentistry, University of Connecticut Health Center, Farmington, CT
06030.
Abbreviations used: BSA, bovine serum albumin; ELISA, enzymelinked immunosorbent assay; GERL, Golgi-endoplasmic reticulumlysosome; IgG, immunoglobulin G; IgM, immunoglobulin M; mAb,
monoclonal antibody; Mr, relative mobility; PAGE, polyacrylamide
gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium
dodecyl sulfate; TBS, Tris-buffered saline.
308
K. TAKANO ET AL.
A
B
C
D
E
F
k Da
- 1 1 0
-
a 4
-
7 7
-
4 7
-
3 3
-
2 4
Fig. 1. Analysis of saliva and agglutinin by electrophoresis and Western blotting. Samples were separated by SDS-PAGE (10% acrylamide) and either silver stained (A,B) or blotted onto nitrocellulose
(C-F). The relative mobility of molecular weight markers is shown on the right. A, parotid saliva, silver
stain; B, purified agglutinin, silver stain; C , Western blot of parotid saliva reacted with mAb 303; D,
Western blot of saliva reacted with polyclonal antibody to denatured agglutinin; E, Western blot of saliva
reacted with polyclonal antibody to amylase; F, Western blot of purified agglutinin reacted with mAb
303.
The appearance of the granules is clearly dependent
upon their composition, but the biochemical basis for
their complex structural organization is currently
poorly understood.
The acinar secretory granules are the source of most
of the more than 50 proteins present in human saliva,
which include digestive enzymes, antimicrobial factors, and proteins which adhere to and coat the surfaces
of the oral cavity (Ellison, 1979). We have selected two
of these proteins, a-amylase and agglutinin, to investigate specific aspects of protein transport and packaging in human salivary acinar cells. a-Amylase represents a family of proteins, some of which are
glycosylated, t h a t are secreted by the salivary glands of
most species and cleave starch to limit dextrins (Merritt and Karn, 1977). The agglutinin is a high molecular weight acidic sialoglycoprotein that binds to and
aggregates specific oral streptococci (Hay et al., 1971;
A
GS
ICS
IG
L
M
SD
SG
A bbreuiations
acinar cells
Golgi saccules
lateral cell surface
immature secretory granule
lumen
mucous cells
striated duct
secretory granule
Kashket and Donaldson, 1972; Malamud and Golub,
1981).This agglutinin has been isolated from both parotid and submandibular saliva of humans, and shown
to be distinct from secretory IgA and mucins (Williams
and Gibbons, 1975; Malamud, 1985). The agglutinin
has been purified by both column chromatography (Demuth et al., 1988) and bacterial affinity (Rundegren
and Arnold, 1987). The isolated glycoprotein is approximately 40% carbohydrate and 60% protein (Ericson
and Rundegren, 1983). Glycoproteins with similar
functions have been described in the saliva of rats
(Brack and Reynolds, 1987). We have used mouse polyclonal and monoclonal antibodies (mAbs) directed
against the agglutinin, and a rabbit polyclonal antibody to human salivary amylase, to localize these
secretory proteins by post-embedding immunogold labeling procedures. Our results demonstrate t h a t agglutinin and amylase are differentially distributed within
the secretory granules, and suggest that distinct Golgi
subcompartments may be involved in the transport and
sorting of these two proteins.
MATERIALS AND METHODS
Agglutinin was purified from human parotid saliva
by column chromatography on DEAE-Sephadex and
Sepharose 4B a s previously described (Demuth et al.,
1988) and shown to migrate a s a single band on SDS-
309
SALIVARY AGGLUTININ AND AMYLASE DISTRIBUTION
TABLE 1. Characterization of anti-agglutinin
monoclonal antibodies
mAb
Isotype
no, = 1
td i n h i b i t
143
116
303
IeG,
IgG;
IgM
0% at 20 mM
80% at 0.5 mM
80% at 0 . 5 m M
Titer
(Western blot)
1:lOO
1:1,000
1:500
prepared from the spleens of these mice, and fused
(polyethylene glycol) with SP 2/0 Ag14 myeloma cells.
Approximately 350 clones were screened using 1)a bioassay in which culture media was mixed with saliva
and tested for inhibition of bacterial aggregation (Malamud et al., 1983),and 2) a n ELISA using PVC plates
coated with purified agglutinin and exposed to culture
media and then a second antibody conjugated to peroxidase. Of the original 350 clones, 62 were positive in
both assays. Eight of these have been recloned three
times and seven of these have been characterized with
respect to isotype, titer €or inhibition and ELISA, and
effects of sodium periodate and neuraminidase treatments. Additionally, a polyclonal antibody to denatured agglutinin eluted from SDS electrophoretic gels
was prepared in mice.
Light Microscopic lmmunohistochemistry
Parafin-embedded formalin or Bouin’s fixed tissues
from the Surgical Pathology file of either The Hospital
of the University of Pennsylvania or the American Oncologic Hospital were used. Immunoreactivity was detected using the preformed avidin-biotin-peroxidase
complex procedure (Vectastain ABC Kit, Vector Laboratories). Sections were incubated for 15 min in 0.3%
H,Oz in methanol to block endogenous peroxidases.
Following a wash in PBS, slides were incubated sequentially in normal horse serum for 20 min, with appropriately diluted mAbs for 30 min at room temperature, biotinylated anti-mouse immunoglobulin (1:200)
and avidin-biotin-peroxidase complex. Sections were
incubated with 0.05% diaminobenzidine (Sigma Chemical Co.) with 0.01% H20, for 5 min and counterstained
with hematoxylin. Mouse myeloma-P3 supernatant
was used as a negative control.
Tissue Preparation for Electron Microscopy
Fig. 2. Light microscopic immunolocalization of agglutinin in human salivary glands with mAb 303. A-C: Submandibular gland. D:
parotid gland. Serous acinar (A) and demilune cells (arrowheads in A
and B) are labeled, but no reaction is seen in mucous cells (M). The
luminal and basal portions of the striated ducts (SD) are also stained.
x 435.
PAGE with a subunit molecular weight of approximately 400,000 Daltons (Fig. 1).
Preparation of Antibodies to Salivary Agglutinin
BALBic mice were immunized with purified agglutinin in Freund’s adjuvant. A single cell suspension was
Normal human salivary gland tissues were obtained
from surgical specimens (Brown et al., 1985).Promptly
after surgical removal, the tissue specimens were cut
into small pieces (1mm3) in cold fixative solution. Fixatives used were 3% (para)formaldehyde plus 0.1% glutaraldehyde, 1% glutaraldehyde, and 4% (para)formaldehyde in 0.05 or 0.1 M phosphate or cacodylate buffer,
pH 7.4. Total fixation time was approximately 1 hour
for glutaraldehyde-containing fixatives, and 4 to 6
hours for formaldehyde-containing fixatives. The tissue samples were rinsed in 0.1 M phosphate or cacodylate buffer, pH 7.4, with 5-7% sucrose, then dehydrated and embedded in LR White resin (London Resin
Co.) or EMbed 812 (Electron Microscopy Sciences)
without osmium postfixation. Some tissue pieces from
samples fixed in 1%glutaraldehyde were postfixed in
1% OsO, and processed for conventional morphologic
evaluation. Additionally, some fixed and embedded tissue samples were generously provided by Professor A.
Riva, University of Cagliari, Italy. Semithin sections
310
K. TAKANO ET AL.
Fig. 3. Acinar secretory granules of human submandibular gland,
immunolabeled with mAb 303 and 15 nm protein A-gold. In A, the
agglutinin is restricted to the peripheral electron-lucent region of the
granules. Only a few particles are present over the dense cores and
the areas of medium density. x 28,000. In B, labeling of the medium
density areas of the granules with mAb 303 is heavy, but the electrondense cores are unlabeled. Labeling is also seen on the cell membrane
a t the luminal (L) and lateral (ICS) surface. x 21,000.
Fig. 4. Submandibular acinar cell granules immunolabeled with
polyclonal anti-agglutinin antibody and 15 nm protein A-gold. The
labeling pattern is identical to that seen with mAb 303: gold particles
are concentrated over regions of low and medium electron density in
the secretory granules, and are present on the luminal (L) and lateral
(ICS) cell surfaces. x 30,000.
were stained with toluidine blue and appropriate areas
were selected for thin sectioning. Ultrathin sections
were collected on Formvar-coated or bare nickel grids
for immunostaining, or on copper grids for routine observation.
Electron Microscopic lmmunocytochemical Procedures
The thin sections were immunolabeled using the protein A-gold method, modified from Roth et al. (1978)
and Bendayan et al. (1980). The grids were floated se-
SALIVARY AGGLUTININ AND AMYLASE DISTRIBUTION
311
lase was visualized with 5 nm gold particles and agglutinin with 15 nm particles.
Immunocytochemical controls included 1)incubation
of sections with primary antibody which had been preabsorbed by incubation with a n excess of the antigen
overnight a t 4°C; 2) incubation of the sections with
non-immune immunoglobulins or a n irrelevant mAb of
the IgG or IgM class instead of the primary antibody;
and 3) omission of the primary antibody andior the
secondary antibody from the staining sequence.
RESULTS
Characterizationof Antibodies
The characterization of the three mAbs utilized for
immunolabeling is shown in Figure 1 and Table 1.
Western blots of human parotid saliva and purified agglutinin (Fig. 1)demonstrated staining of a single band
migrating with a n approximate Mr of 400,000 Daltons.
Some diffuse staining of smaller sized components was
also noted. Monoclonal antibodies 303 and 116 appeared to recognize carbohydrate epitopes based on the
sensitivity of the ELISA to inhibition by sodium periodate (Table l).None of the mAbs required sialic acid
for recognition, as neuraminidase treatment did not
decrease reactivity (data not shown). In addition, isotyping revealed that mAbs 143 and 116 are IgGs,
whereas mAb 303 is a n IgM.
Western blots of saliva incubated with the polyclonal
antibody to the denatured agglutinin revealed a single
reactive band (Fig. 1).
Fig. 5. Submandibular acinar granules immunolabeled with antiTo test whether any of the three mAbs recognize a
amylase antibody and 15 nm protein A-gold. Amylase is present in
common epitope, the technique of Friguet et al. (1983)
the dense core and the areas of medium density, whereas the regions
was used. Each antibody was titered in a n ELISA using
of low electron density are unlabeled. x 22,000.
a fixed concentration of antigen (agglutinin). Selecting
the antibody concentration giving one-half maximum
optical density, mAbs were then tested in combination.
quentially on small drops (25-30 PI) of PBS for 5 min; These experiments indicated that mAbs 303 and 116
1% BSA in TBS for 30 min; and either the polyclonal recognized the same epitope.
In an attempt to identify the carbohydrate epitope
anti-agglutinin or one of the monoclonal anti-agglutinins diluted in 0.02 M Tris-HC1, pH 7.4, with 0.5 M recognized by mAbs 303 and 116, a series of known
NaCl and 0.1% Tween 20 for 60 min at room temper- glycolipids was separated on TLC plates and reacted
ature. In some cases incubation with the mAb was done with the mAbs and also with a panel of mAbs with
overnight at 4°C. After rinsing with the Tris-NaC1- known specificities (Magnani et al., 1982). Results of
Tween buffer, the grids were incubated with the appro- these studies indicated that mAbs 303 and 116 recogpriate unlabeled secondary antibody (affinity-purified nized the Y epitope (Blaszczyk-Thurin et al., 1987).
rabbit anti-mouse IgG or goat anti-mouse IgM, Orga- This was confirmed by demonstrating that these mAbs
non Teknika-Cappel) diluted in TBS for 60 min; rinsed agglunitated latex beads containing Y-BSA conjugates
in TBS; and incubated with 10 or 15 nm diameter pro- but not beads with X, Lewis a, or Lewis b conjugates.
Figure 1also shows the reaction of anti-amylase antein A-gold complex (Janssen Life Science Products)
diluted 1 : l O in TBS for 30 min. The grids were rinsed tibodies (Sigma) on Western blots of human parotid
with TBS and finally with distilled water before being saliva. A double band migrating with a n Mr of approxdried and stained with uranyl acetate and lead citrate. imately 60,000 Daltons was labeled. Presumably, the
Immunolabeling for a-amylase was performed in a two bands correspond to the glycosylated and non-glysimilar manner, using a polyclonal antibody to human cosylated forms of human salivary amylase (Keller et
salivary amylase (Sigma) diluted in TBS, followed by al., 1971).
rinsing with TBS and visualization with protein ALight Microscopic lmmunohistochemicalLocalization of
gold.
Salivary Agglutinin
Double labeling for a-amylase and the agglutinin
Sections of five human submandibular glands were
was performed by the two-sided method of Bendayan
(1982). One side of the section was incubated for 60 min incubated with varying concentrations of the three
with anti-amylase antibodies, and the bound IgG was mAbs described above. In all cases, staining was seen
localized with 15 nm protein A-gold complex. The grid in serous acinar (Fig. 2A-C) and demilune cells (Fig.
was rinsed and dried, and the other side was incubated 2A,B), intercalated duct cells, and striated duct cells
with mAb 303, goat anti-mouse IgM, and finally 5 nm (Fig. 2C). In the striated ducts, the labeling appeared
protein A-gold (Janssen). In some experiments, amy- strongest at the luminal surface and in the infranu-
312
K. TAKANO ET AL.
Fig. 6. Double labeling of acinar secretory granules with mAb 303
and anti-amylase antibody. Agglutinin (5 nm gold particles) is
present in areas of low electron density, and amylase (15 nm gold
particles) is present mainly in the dense regions. Some reactivity for
amylase is also seen along with agglutinin in areas of low density
(arrowheads). ~46,000.
clear region. No labeling of mucous acinar cells occurred. In the parotid gland (Fig. 2D), which is a pure
serous gland, a similar distribution was observed for
the agglutinin in the acini and ducts.
al., 1974). In the present study, two or three regions of
differing electron density were observed in the granules of most cells. Labeling for the agglutinin with either mAb 303 (Fig. 3) or the polyclonal anti-agglutinin
antibody (Fig. 4) was consistently observed in the regions of lowest electron density, which usually were
located peripherally in the granules. The dense corelike structures, often positioned eccentrically, were unlabeled, whereas regions of medium density showed a
variable labeling intensity. With anti-amylase antibody, the opposite labeling pattern was observed (Fig.
5). The dense cores labeled most intensely, whereas
regions of lowest density were unlabeled. Again, areas
of moderate density showed a variable labeling intensity.
Sections labeled with both anti-amylase antibody followed by 15 nm protein A-gold, and mAb 303 followed
by 5 nm protein A-gold, to simultaneously localize both
proteins (Fig. 61, showed a labeling pattern similar to
that seen in singly labeled preparations. Low density
regions were labeled almost exclusively with 5 nm gold
particles, and the dense cores were labeled only with 15
nm gold particles, indicating the presence of agglutinin
and amylase, respectively, in these two regions. In
some granules, both small and large gold particles, in
variable proportions, were present over low to moderately dense regions (arrowheads, Fig. 6), showing a
mixture of the two proteins in these areas. An identical
lmmunogold Labeling of Submandibular Gland Acinar Cells
In acinar cells of the human submandibular gland,
immunolabeling for the agglutinin and for amylase
was present in the Golgi region and over the secretory
granules. Additionally, some of the anti-agglutinin
mAbs also labeled the apical and basolateral membranes. Labeling with the mAbs was most intense with
mAb 303; labeling was less intense with mAb 116, but
the distribution was similar. With mAb 143, which recognizes a polypeptide epitope, labeling of the granules
and Golgi apparatus was weak and no reactivity was
observed on the cell membranes. The present report
focuses on the labeling of the Golgi apparatus and
secretory granules of the acinar cells; a complete description of the labeling patterns of the various cell
types present in the human salivary glands obtained
with the three mAbs will be communicated separately.
Secretory granules
The secretory granules of human submandibular acinar cells typically contain internal substructures of
variable morphology and electron density (Tandler and
Erlandson, 1972; Riva and Riva-Testa, 1973; Riva et
SALIVARY AGGLUTININ AND AMYLASE DISTRIBUTION
313
Fig. 7. Double labeling of Golgi apparatus with mAb 303 (5 nm
particles) and anti-amylase antibody (15 nm particles). Large gold
particles demonstrating amylase are present over the Golgi saccules
(GS) and the dense core of a small forming granule (arrow).Only a few
small particles representing agglutinin are seen over the electronlucent area of the forming granule and in the trans saccules (arrowheads). x 63,000.
Fig. 8. Double labeling of Golgi apparatus. Agglutinin reactivity (5
nm particles) is seen in small tubules (arrowheads) and immature
secretory granules (IG) a t the trans face, in regions of low density in
the mature secretory granule (SG), and in a large tubular structure
(arrow) at the left of the micrograph. Amylase reactivity (15 nm particles) is low but gold particles are seen over the Golgi saccules (GS),
immature granules and the dense regions of the mature granule.
x 52,000.
distribution of the two proteins was seen in preparations in which the sizes of the gold particles were reversed (data not shown).
with mAb 303 was restricted to structures in the trans
Golgi region. These included immature secretory granules (Figs. 7-9); vesicles, tubules, and cisternae with
morphological characteristics of the trans Golgi network (Griffths and Simons, 1986) or GERL (Novikoff,
1976; Hand and Oliver, 1977a,b) (Figs. 8-11); and occasionally the trans Golgi saccule (Fig. 7). The remainder of the Golgi saccules were unlabeled. Occasional
Golgi apparatus
The distribution of the two secretory proteins in the
Golgi region was most clearly demonstrated in doublylabeled sections. Immunolabeling for the agglutinin
314
K. TAKANO ET AL.
Fig. 9. Double labeling of Golgi apparatus. Amylase (15 nm particles) is seen in the Golgi saccules (GS) and the dense core of one
forming granule. Agglutinin reactivity (5 nm particles) is seen in the
same forming granule, in elements of the trans Golgi network (GERL)
(arrow), and in a small tubular or vesicular structure a t the cis face
(arrowhead). x 53,000.
tubular and/or small spherical bodies which labeled
only with mAb 303 were also present. These structures
were often seen in the Golgi region (Fig. 8), where they
were associated with both faces of the Golgi apparatus,
and also in peripheral regions of the cytoplasm, sometimes near the basolateral membrane (Fig. 12A,B).
Similar vesicular structures near the basolateral membrane could be identified in unincubated sections of
specimens postfixed in OsO, (Fig. 12C,D). In the absence of labeling with mAb 303, however, it was not
possible to determine if these vesicles were the same
structures as those seen in immunolabeled sections.
Immunolabeling for amylase, in contrast, was seen
over the Golgi saccules (Figs. 7-10), but only rarely
over structures in the trans Golgi region labeled with
mAb 303. Immature secretory granules, especially
those showing condensations of dense material (Figs.
7-9), were labeled with the antibody to amylase. When
these forming granules also were labeled with mAb
303, small gold particles indicating the presence of the
agglutinin were located over the electron-lucent regions, whereas large particles representing amylase
were present over the dense core (Figs. 7, 8).
lmmunocytochemical controls
Omission of the primary antibodies from the staining
sequence, or substitution of non-immune immunoglobulins for the primary antibodies, essentially eliminated
the binding of gold to the sections for both agglutinin
and amylase. Preabsorption of the antibodies with
their respective antigens also greatly reduced labeling
of the secretory granules.
DISCUSSION
Amylase and agglutinin, two secretory proteins of
human salivary acinar cells, are both packaged in the
same secretory granules. The double labeling results
clearly demonstrate, however, that within individual
secretory granules, these two content proteins are differentially distributed. Amylase is concentrated in the
denser portions of the granule content, whereas agglu-
tinin is found in the less dense regions. These findings
are consistent with the results of previous immunolabeling studies of salivary glands of various species
showing preferential localization of secretory proteins
in specific regions of the granules. For example, amylase, lysozyme and proline-rich proteins are concentrated in the dense regions of the granules of serous
cells (Ichikawa et al., 1989; Machino et al., 1986; Kousvelari et al., 1982), neonatal and adult secretory proteins show a differential distribution in certain granules of the postnatal rat submandibular gland (Moreira
et al., 19911, and mucin is located in the electron-lucent
regions of adult rat submandibular mucous cells
(Moreira et al., 1989). Differential localization of secretory proteins has also been observed in granules of
some endocrine cells (Ravazzola and Orci, 1980; Fumagalli and Zanini, 1985; Bendayan, 1989). Although
the distribution of amylase and agglutinin was correlated with the electron density of the granule content,
some variability was noted. Thus, both proteins were
often found in regions of moderate density. Also, the
size, shape, and relative electron density of the different intragranular regions was found to vary among
granules. These factors may be responsible for the difference in our results and those of Machino et al.
(1986), who reported a relatively homogeneous distribution of amylase in human parotid and submandibular acinar cell granules. In contrast to the distribution
of the A, B, and H blood group antigens in human
salivary glands (Nakajima et al., 1987), amylase and
agglutinin reactivities were observed in the Golgi apparatus and immature secretory granules, as well as
the mature granules.
Several underlying questions remain unanswered.
For example, why do secretory granules of some cells,
such as rat parotid (Ball e t al., 1988; Hand and Ball,
1988) and pancreatic acinar cells (Bendayan et al.,
1980), exhibit a relatively homogeneous distribution of
several secretory proteins, whereas those of the human
salivary glands display such a heterogeneous distribution of their content? What is the molecular basis for
the segregation of different proteins within the granule? Is it due to the specific physical-chemical properties of individual content proteins, resulting in self- or
coaggregation (Burgess and Kelly, 1987; Stoller and
Shields, 1989),or to other components involved in condensation and packaging, such as sulfated macromolecules (Palade, 1975), calcium (Clemente and Meldolesi,
19751, specific binding proteins (Chung et al., 1989), or
perhaps a n internal reticular component involved in
organization of the granule content (Ermak and Rothman, 1978)?
I t is conceivable that the segregation of the granule
contents is a n artifact of chemical fixation, and t h a t a
homogeneous distribution of secretory proteins exists
in vivo. Several observations argue against such a simple explanation, however. The structure of the granules appears relatively similar in several studies of human salivary glands (Tandler and Erlandson, 1972;
Riva and Riva-Testa, 1973; Riva et al., 1974), despite
varied fixative formulations and fixation protocols. It
seems unlikely that the extremely elaborate substructural organization found in granules of many species
(Tandler and MacCallum, 1972) could be a result of
fixation-induced molecular rearrangements, without
SALIVARY AGGLUTININ AND AMYLASE DISTRIBUTION
Figs. 10, 11 : Double labeling of Golgi apparatus. Amylase (15 nm particles) is present in the Golgi
saccules (GS) and secretory granules (SG). In Figure 10, agglutinin reactivity (5 nm particles) is seen in
several tubular elements of the trans Golgi network (GERL) (arrowheads). x 69,000. In Figure 11, an
extensive tubular network a t the trans face contains agglutinin reactivity (5 nm particles). Agglutinin
is also seen in the secretory granules. x 90,000.
315
316
K. TAKANO ET AL.
Fig. 12. A , B Double labeling for agglutinin and amylase. Small
vesicles (arrowheads) labeled only for agglutinin (5 nm particles) are
present near the lateral membrane. In A, agglutinin reactivity is also
seen on the cell surf-ace (arrow). C , D Small vesicles (arrowheads) are
seen near the lateral membrane of acinar cells in Os0,-postfixed tissue. A, x 63,000; B, x 67,000; C, x 86,000; D, x 58,000.
the presence of a variety of intermediate forms where
stabilization occurred before the rearrangements were
completed. Finally, secretory granules exhibiting complex intragranular substructures andlor regions of
variable density, a s well as a heterogeneous distribution of amylase have been described in rapidly frozen,
freeze-substituted gerbil sublingual and parotid glands
(Ichikawa and Ichikawa, 1987; Ichikawa e t al., 1989).
It is possible that the differential distribution of
amylase and agglutinin within the granules results
from the apparently separate pathways these two proteins travel in reaching the forming secretory granules. Agglutinin appears to reach the granules through
the trans Golgi network (GERL); agglutinin reactivity
was present in the trans saccules and in elements of the
trans Golgi network. Although agglutinin reactivity
was not seen in the cis saccules, this is consistent with
the specificity of mAb 303 for a fucose-containing carbohydrate epitope, because fucose is incorporated into
glycoproteins in the trans Golgi saccules (Bennett,
1984; Roth, 1987). In contrast, the route followed by
amylase is presently unclear. Amylase reactivity was
found in the Golgi saccules, but not in structures identifiable as part of the trans Golgi network (GERL).
Both amylase and agglutinin were present in the earliest identifiable granules forming at the trans face,
and amylase appeared to be associated with the dense
core a s i t initially began to condense.
The trans Golgi network is generally regarded as the
site of sorting of secretory, lysosomal, and membrane
proteins (Griffiths and Simons, 1986) and the point of
divergence of the regulated and constitutive pathways
(Burgess and Kelly, 1987). In some cells, enzyme- and
immunocytochemical studies have failed to demonstrate secretory proteins (including amylase) in the
trans Golgi network (Hand and Oliver, 1977a; Geuze et
al., 1979; Bendayan, 1984,1989),despite its continuity
with and apparent involvement in secretory granule
formation. In other cells, a s in the present study, secretory proteins are clearly present in elements of the
trans Golgi network (Geuze et al., 1984, 1985; Tooze
and Tooze, 1986). These observations suggest t h a t in
different cells, or even within a single cell, different
routes may exist between the trans Golgi saccule and
the forming granules, and/or that sorting of regulated
proteins may occur at more than one site in the secretory pathway.
A complicating factor in assessing the labeling of the
trans Golgi compartments with mAb 303 is the apparent reactivity of this antibody with a shared carbohydrate epitope on one or more membrane glycoproteins
located on the basolateral and luminal surfaces of the
acinar cells. Consistent with this interpretation is the
fact that mAb 116, which recognizes the same carbohydrate epitope, also labels the cell membrane,
whereas mAb 143, with specificity for a polypeptide
epitope, does not. The trans Golgi region probably is
the site of sorting of the secretory agglutinin from
these putative membrane glycoproteins, and at least
part of the label in the trans Golgi region may be attributed to the latter. However, the abundance of agglutinin in the secretory granules, and the presumably
higher turnover rate of the granules compared with
that of the cell surface components recognized by mAb
303, suggest that most of the reactivity seen in the
trans Golgi region must be agglutinin.
Interestingly, small vesicles and tubules labeled
with mAb 303, but not with anti-amylase antibody,
were observed at both faces of the Golgi apparatus and
frequently near the cell membrane. It is tempting to
suggest that these structures are the morphological
correlates of the constitutive secretion pathway in
these cells, carrying membrane glycoproteins to the
cell surface. On the other hand, i t is equally possible
that these vesicles and tubules are part of the endocytic
apparatus of these cells, and contain cell membrane
glycoproteins destined for degradation in the lysosomal
system. We did not observe any morphologically identifiable lysosomes labeled with mAb 303, but this may
indicate that the epitope had already been degraded.
Studies with additional markers, such a s antibodies to
other cell membrane glycoproteins or to lysosomal
membrane proteins such as lgp 120 (Lewis et al., 1985)
or endolyn 78 (Croze et al., 1989), may help to resolve
the nature of these vesicles and tubules.
SALIVARY AGGLUTININ AND AMYLASE DISTRIBUTION
317
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distributions, amylases, secretory, granules, apparatus, agglutinin, human, acinar, cells, gland, differential, salivary, golgi
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