 |
|
High ß-Galactosidase and Ganglioside GM1 Levels in the Human Parotid Gland
Nakisa Nowroozi, PhD;
Toshitsugu Kawata, DDS, PhD;
Peixin Liu, PhD;
Dale Rice, MD;
Joseph H. Zernik, DMD, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:1381-1384.
ABSTRACT
| |
Background Ganglioside GM1 is a membrane glycolipid typical of nerve
cell membranes, where it partakes in neurotransmitter release and is catabolized
by the lysosomal ß-galactosidase (GM1ase) (EC 3.2.1.23). After demonstrating
a novel degenerative disease of the parotid gland in mice deficient in GM1ase,
mimicking the human storage disease GM1 gangliosidosis, we studied
GM1ase and ganglioside GM1 content in the human parotid glands.
Study Design Levels of GM1ase and ganglioside GM1 were determined in samples
of parotid tissues and neighboring muscle (as a negative control) for 3 subjects.
Tissues were also processed for histochemical demonstration of GM1ase.
Results The mean specific activity of GM1ase was more than 6-fold higher in
the healthy human parotid tissues (1.4 ± 0.5 nmol of 4-methylumbelliferone
per minute per milligram of protein) relative to the neighboring muscle tissue
(0.23 ± 0.07 nmol of 4-methylumbelliferone per minute per milligram
of protein). Activity of GM1ase was histochemically localized mainly to striated
duct and acinar cells of the parotid gland. Ganglioside GM1 content
in the parotid gland was on average 30-fold higher relative to muscle.
Conclusions Our results are consistent with previous findings reported in the mouse
and the rabbit, and probably reflect a general property of the mammalian parotid
glands. The novel mechanism we previously proposed for the mouse parotid saliva
secretion, mimicking neurotransmitter release in ganglioside GM1–containing
nerve cells, is probably applicable also to the human parotid gland. Similarly,
the human parotid gland is probably also severely affected in GM1
gangliosidosis.
INTRODUCTION
GANGLIOSIDE GM1 is a membrane glycolipid, typical of nerve
cell membranes, where it partakes in neurotransmitter release and is catabolized
by the lysosomal ß-galactosidase (GM1ase) (EC 3.2.1.23).1
Previous studies from our laboratory demonstrated a surprisingly high level
of GM1ase and ganglioside GM1 in the parotid glands of mice2 and a novel degenerative disease of the parotid in
knockout mice deficient in GM1ase3 mimicking
the human storage disease GM1 gangliosidosis.3-4
This autosomal recessive disorder, resulting from GM1ase deficiency, primarily
affects the brain, where storage of ganglioside GM1 is seen primarily
in gray-matter nerve cells.1
In the present study, we analyzed human parotid tissues using biochemical
and histochemical approaches. Findings reported herein demonstrate that high
levels of GM1ase activity and ganglioside GM1 content, previously
shown in the mouse, are properties shared by the human parotid glands.
MATERIALS AND METHODS
TISSUE SAMPLES
Surgical discard tissues were assayed from 3 subjects (1 woman and 2
men, aged 28 to 50 years) undergoing excision of pleomorphic adenomas of the
parotid gland. During surgery, neighboring margins of healthy parotid and
muscle tissues were also excised. The samples we used included these healthy
parotid and muscle tissues, as identified by the surgeons and verified by
results of histological examination.
PROTEIN ASSAYS
Tissue samples were rinsed in ice-cold phosphate-buffered saline solution
and homogenized to a concentration of 5 mg/mL of wet tissue weight in a buffer
containing 0.01M Tris (pH, 6.7), 0.1% sodium dodecyl sulfate, and serine protease
inhibitor (Pefabloc, St Louis, Mo) at a concentration of 0.1 mg/mL. Protein
assays were performed on homogenates using a commercially available assay
(Bio-Rad Laboratories Inc, Hercules, Calif) and bovine serum albumin as a
standard.5
GM1ase ENZYME ASSAYS
Three different serial (2-fold) dilutions of each homogenate were tested
for ß-galactosidase activity under acidic conditions by means of a fluorometric
assay using 4-methylumbelliferyl– ß-D-galactopyranoside (4-MU–ß-gal)
(Sigma-Aldrich Corp, St Louis, Mo) in a solution containing 2mM substrate
and 0.1M sodium acetate buffer (pH, 4.4), 0.2M sodium chloride, and 0.25%
bovine serum albumin. Samples were incubated for 30 minutes at 37°C. These
reactions were stopped by adding a 10-fold higher volume of 0.2M borate buffer
(pH, 9.8). The liberated 4-MU was measured using spectrofluorometery with
excitation at 366 nm (10-nm slit) and emission at 446 nm (20-nm slit) compared
with 4-MU standards.
HISTOLOGICAL AND HISTOCHEMICAL ANALYSIS
For microscopic histochemical study, a modification of the method of
Sanes et al6 was performed under acidic conditions.2 The tissues were prefixed for 2 hours in 10% formaldehyde,
made permeable at 37°C for 2 hours in 2mM magnesium chloride, 0.01% sodium
deoxycholate, 0.02% NP-40, 8.25-mg/mL potassium ferrocyanide, and 10.5-mg/mL
potassium ferricyanide, and stained overnight at 37°C in 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
(x-gal) (Research Organics Inc, Cleveland, Ohio) in 0.1M sodium acetate (pH,
4.4), 0.2M sodium chloride, and 0.25% bovine serum albumin. Poststain fixation
was performed in 10% neutralized buffered formalin for 1 hour, with consequent
dehydration in a graded series of ethanol to 100%. Tissues were cleared in
xylene, infiltrated, and embedded in paraffin, and 5-µm sections were
prepared using a Reichardt-Jung microtome (Leica Microsystems Nussloch GmbH,
Nussloch, Germany).
GANGLIOSIDE ANALYSIS
Our method for quantitative analysis of ganglioside GM1 is
based on a modification of the method described by Svennerholm.7
Parotid and control tissue samples (wet tissue weight, 250 mg) were homogenized,
and their total gangliosides were extracted based on differential solubility
in a mixture of chloroform, methanol, and water. The amphipathic nature of
gangliosides makes such an extraction method possible. The order of addition
of the solvents and the exact volume ratios are essential for effective extraction
of the gangliosides and effective elimination of the various hydrophilic and
hydrophobic contaminants.
The tissue was first homogenized in ice-cold distilled water (3 x
volume of tissue weight), then methanol (10.6 x volume of tissue weight)
and finally chloroform (5.3 x volume of tissue weight) were added. Solids
were pelleted by means of centrifugation, and the pellet was reextracted using
the chloroform-methanol-water mixture to ensure maximal extraction of gangliosides
from the tissue. Distilled water (0.173 x volume of tissue weight) was
added to the combined supernatants containing the gangliosides and other contaminants.
The addition of the water caused the separation of the original extraction
solution into 2 phases, and the gangliosides partition into the aqueous phase.
The 2 phases were separated and each reextracted, and the aqueous phase was
further purified using reverse-phase chromatography (Sep-Pak Plus C; Waters
Corp, Milford, Mass). Final elution of the column was performed in methanol,
and the purified ganglioside samples were then lyophilized, dissolved in a
small volume (7 µL) of chloroform and methanol (1:2), and applied to
thin-layer chromatography (TLC) silica gel 60 plates (EM Science, Gibbstown,
NJ). The gangliosides were resolved by means of TLC in a mixture of chloroform,
methanol, and 10mM potassium chloride (55:45:10, vol/vol). The addition of
potassium chloride altered the mobility of gangliosides and improved their
resolution. The ganglioside bands were visualized using a combination of resorcinol,
hydrochloric acid, and copper ion as a staining agent, which requires activation
by heating at 120°C for 20 minutes. The ganglioside bands appeared violet-blue
while neutral, and sulfated glycosphinogolipids appeared yellow-brown. Pure
ganglioside GM1 (2 µg per lane) (Sigma-Aldrich Corp) was
used as a migration marker and a quantitative standard. The bands of interest
were quantitated by means of densitometric measurements. After correcting
for the background for each plate, the net optical density measurements for
each tissue were divided to the standard measurement on the same plate.
RESULTS
Ganglioside GM1 is a membrane glycolipid typical of nerve
cells, catabolized in the lysosomes of such cells by GM1ase. Previous studies
demonstrated a surprisingly high level of GM1ase and ganglioside GM1 in the parotid glands of mice, and a novel degenerative disease of
the parotid gland in mice deficient in GM1ase, mimicking the human storage
disease GM1 gangliosidosis.3-4
Our results determined GM1ase-specific activity in healthy human parotid tissues
of 3 subjects. Muscle, a tissue previously reported as being low in GM1ase
activity, as are almost all mammalian and human tissues, was used as our negative
control. Our data demonstrate that mean GM1ase-specific activity in the healthy
human parotid samples was significantly and substantially (>6-fold) higher
than that in healthy muscle samples (P<.001) (Figure 1).
|
|
|
|
Figure 1. Acidic ß-galactosidase–specific
activity in human tissue extracts. Specific activity in neighboring muscle
tissue is significantly lower than that found in healthy human parotid tissue
(P<.001). 4-MU indicates 4-methylumbelliferone.
|
|
|
We used the x-gal substrate under acidic conditions to histochemically
demonstrate GM1ase activity as blue staining in the parotid glands (Figure 2A-B), whereas such activity was undetectable
in muscle (Figure 2C-D). High levels
of x-gal staining, indicative of GM1ase activity, were localized to parenchymal
cells of the parotid gland. Highest-intensity staining was found in the apical
cytoplasm of striated duct cells and lower-intensity staining was found in
acinar cells of parotid sections, whereas findings in the muscle appeared
negative. In the acinar cells, reaction products appear primarily in the basal
cytoplasm. Endothelial cells also seem to show weak reaction products.
|
|
|
|
Figure 2. Histochemical ß-galactosidase
activity in human tissue sections containing ganglioside GM1. Activity
of GM1ase (lysosomal ß-galactosidase) is demonstrated as blue (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
[x-gal]) staining. A, Parotid gland (hematoxylin-eosin, original magnification
x100). B, Parotid gland (x-gal staining, original magnification x100).
Arrow indicates the striated ducts; asterisk, acini. C, Muscle (hematoxylin-eosin,
original magnification x100). D, Muscle (x-gal staining, original magnification
x100).
|
|
|
We further assayed ganglioside GM1 content of the human parotid
glands using the TLC technique (Figure 3).
When using this method, ganglioside GM1 in the human parotid gland
appears as a dark stain consistent with that of an authentic sample of ganglioside
GM1. In contrast, no ganglioside GM1 stain is observed
in the muscle sample. Scanning of TLC plates indicate that ganglioside GM1 levels in the human parotid gland are at least 30-fold higher than
that in human muscle (P<.001). Our results demonstrating
high levels of ganglioside GM1 and GM1ase in the human parotid
gland are consistent with previous results in rabbit and mouse.3-4
Therefore, these findings may be a general feature of the mammalian parotid
gland.
|
|
|
|
Figure 3. Thin-layer chromatography of ganglioside
GM1 in tissue extracts. Ganglioside GM1 content in the
healthy human parotid tissue (PAR) is compared with muscle (MUS), and ganglioside
GM1 standard (GM1). Ganglioside GM1 is on
average 30-fold higher in human parotid tissue compared with muscle (P<.001).
|
|
|
COMMENT
Previous studies have examined knockout mice2
deficient in GM1ase8-9 to study
the role of this enzyme in the salivary glands.3-4
The results demonstrated ganglioside GM1 accumulation in the parotid
glands of deficient mice as large (diameter, >10 µm) storage vacuoles
in the parotid gland (but not the submandibular or sublingual glands). The
histochemical staining of these storage vacuoles with combined fluorescein
isothiocyanate and cholera toxin, which binds with high affinity and specificity
to ganglioside GM1, confirmed the storage of ganglioside GM1 in these vacuoles.
These results suggest that ganglioside GM1 is the physiologic
substrate for GM1ase in the parotid glands of mice as previously reported
in the nerve cells.1 Therefore, our working
hypothesis is that ganglioside GM1 and GM1ase are important in
the secretory function of the parotid gland, similar to their function in
neurotransmitter release in nerve cells.10-11
Activity of GM1ase in acinar cells of the parotid gland is most likely related
to lysosomal metabolism of ganglioside GM1 and membrane turnover
in conjunction with the secretory exocytosis/endocytosis cycle.2-4
Thus, our proposed model for the role of ganglioside GM1 and GM1ase
in mouse parotid secretion and membrane turnover mechanisms3-4
may apply to human parotid glands as well (Figure 4).
|
|
|
|
Figure 4. A schematic model for the role
of ganglioside GM1 and lysosomal ß-galactosidase (GM1ase)
in parotid secretion and membrane turnover. Round dots indicate glycoproteins;
daggers, ganglioside GM1; SV, secretory vesicle; END, endosome;
LYS, lysosome; and NUC, nucleus.
|
|
|
It is not clear what the functions of ganglioside GM1 and
GM1ase are in duct cells. These cells also exhibit active membrane recycling.
According to Hand et al,12 the endocytosis/exocytosis
cycle in parotid duct cells is primarily related to secretion and reabsorption
of certain electrolytes, principally by the striated and excretory ducts,13 and to secretion of proteins and glycoproteins, mainly
by the intercalated and striated ducts,14 which
are important functions of these gland components. Thus, ganglioside GM1 and GM1ase probably participate in essential subcellular membrane-recycling
processes in acinar and duct cells of the parotid gland. However, their specific
roles in acinar and duct cells of the human parotid glands in health and disease
require further investigation.
Our model for mouse parotid secretion (Figure 4) is based on the model presented by Sandhoff and Van Echten15 for ganglioside GM1 subcellular turnover
and metabolism in nerve cells. We hypothesize that this model3-4
is applicable to the mammalian parotid gland, in conjunction with the principle ß-adrenergic
secretory pathway described by Castle et al.16-17
Our model differs from that of Sandhoff and Van Echten15
by emphasizing the obligatory endocytosis coupled with exocytosis.18 In both models, the final synthesis of secretory
glycoproteins and ganglioside GM1 is completed in the network of
trans–Golgi acinar cells. Ganglioside GM1 is then incorporated
into cell membranes and transported to the plasma membrane. In recent years,
ganglioside GM1 has been established as a major component of caveolae
and caveolaelike domains in cell membranes, which form uncoated membrane pits.19 These domains are essential for vesicular membrane
trafficking mechanisms (endocytosis/exocytosis) in specific nerve cell types.20 Therefore, our model, based on the results of the
GM1ase knockout mice studies,3-4
proposes an important role for ganglioside GM1 in membrane trafficking
and secretory mechanisms in the parotid gland.
Our results predict that human parotid gland is severely affected in
GM1 gangliosidosis, as is the mouse parotid gland in knockout mice
deficient in GM1ase. However, the potential abnormalities of the human parotid
gland in GM1 gangliosidosis have not been reported so far, possibly
as an oversight. Anecdotal evidence suggests high levels of dental caries
in patients with GM1 gangliosidosis, which may be attributed to
deficient salivary secretion. Studies of oral health and parotid gland function
in patients with GM1 gangliosidosis should address the issue.
AUTHOR INFORMATION
Accepted for publication June 11, 2001.
Corresponding author: Joseph H. Zernik, DMD, PhD, School of Dentistry,
University of Southern California, 925 W 34th St, Los Angeles, CA 90089-0641
(e-mail: jzernik{at}hsc.usc.edu).
From the Departments of Orthodontics and Basic Sciences, School of
Dentistry, University of Southern California, Los Angeles (Drs Nowroozi, Liu,
and Zernik); the Department of Orthodontics, Hiroshima University School of
Dentistry, Hiroshima, Japan (Dr Kawata); and the Department of Otolaryngology,
University of Southern California General Hospital (Dr Rice).
REFERENCES
| |
1. Suzuki Y, Sakuraba H, Oshima A. ß-galactosidase deficiency (ß-galactosidosis): GM1-gangliosidosis
and Morquio B. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York, NY:
McGraw-Hill Co; 1995:2785-2824.
2. Nowroozi N, Denny PA, Denny PC, Zernik JH. Two gene products for ß-galactosidase are differentially expressed
in the mouse major salivary glands. J Cranifac Genet Dev Biol. 1998;18:51-57.
3. Nowroozi N, Kim S, Gupta A, Warita H, Zernik J. High levels of GM1-ganglioside ß-galactosidase in the salivary
glands and GM1-like–ganglioside storage in parotids of deficient mice. J Craniofac Genet Dev Biol. 1999;19:41-47.
ISI
| PUBMED
4. Nowroozi N, Kim S, Segawa A, et al. High levels of GM1-ganglioside and GM1 ganglioside ß-galactosidase
in the parotid gland: a new model for secretory mechanisms of the parotid
gland. Otolaryngol Clin North Am. 1999;32:779-791.
FULL TEXT
|
ISI
| PUBMED
5. Bradford M. A rapid sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
FULL TEXT
|
ISI
| PUBMED
6. Sanes JR, Rubenstein JL, Nicholas JF. Use of a recombinant retrovirus to study post-implantation cell lineage
in mouse embryos. EMBO J. 1986;5:3133-3142.
ISI
| PUBMED
7. Svennerholm L. The distribution of lipids in the human nervous system, I: analytical
procedure lipids of fetal and newborn brain. J Neurochem. 1964;11:839-853.
FULL TEXT
|
ISI
| PUBMED
8. Hahn CN, del Pilar Martin M, Schroder M, et al. Generalized CNS disease and massive GM1-ganglioside accumulation in
mice defective in lysosomal acid ß-galactosidase. Hum Mol Genet. 1997;6:205-211.
FREE FULL TEXT
9. Matsuda J, Suzuki O, Oshima A, et al. Beta-galactosidase–deficient mouse as an animal model for GM1-gangliosidosis. Glycoconj J. 1997;14:729-736.
FULL TEXT
|
ISI
| PUBMED
10. Rahmann H, Probst W, Muhleisen M. Gangliosides and synaptic transmission. Jpn J Exp Med. 1982;52:275-286.
PUBMED
11. Thomas PD, Brewer GJ. Gangliosides and synaptic transmission. Biochim Biophys Acta. 1990;1031:277-289.
PUBMED
12. Hand AR, Coleman R, Mazariegos MR, Lustmann J, Lotti LV. Endocytosis of proteins by salivary gland duct cells. J Dent Res. 1987;66:412-419.
FREE FULL TEXT
13. Young JA, Schneyer CA. Composition of saliva in mammalia. Aust J Exp Biol Med Sci. 1981;59:1-53.
ISI
| PUBMED
14. Hand AR. Synthesis of secretory and plasma membrane glycoproteins by striated
duct cells of rat salivary glands as visualized by radioautography after 3H-fucose injection. Anat Rec. 1979;195:317-340.
FULL TEXT
| PUBMED
15. Sandhoff K, Van Echten G. Ganglioside metabolism: topology and regulation. Adv Lipid Res. 1993;26:119-141.
ISI
| PUBMED
16. Castle JD, Jamieson JD, Palade GE. Radioautographic analysis of the secretory process in the parotid acinar
cell of the rabbit. J Cell Biol. 1972;53:290-311.
FREE FULL TEXT
17. Castle JD. Protein secretion by rat parotid acinar cells: pathways and regulation. Ann N Y Acad Sci. 1998;842:115-124.
FULL TEXT
|
ISI
| PUBMED
18. Segawa A, Yamashina S. Roles of microfilaments in exocytosis: a new hypothesis. Cell Struct Funct. 1989;14:531-544.
ISI
| PUBMED
19. Parton RG. Ultrastructural localization of gangliosides: GM1 is concentrated in
caveolae. J Histochem Cytochem. 1994;42:155-166.
ABSTRACT
20. Schnitzer JE, Oh P, Pinney E, Allard J. Fillipin-sensitive caveolae-mediated transport in endothelium. J Cell Biol. 1994;127:1217-1232.
FREE FULL TEXT
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter
What's this?
RELATED ARTICLE
Archives of Otolaryngology–Head & Neck Surgery Reader's Choice: Continuing Medical Education
Arch Otolaryngol Head Neck Surg. 2001;127(11):1403-1405.
FULL TEXT
|
