(Received for publication, November 22, 1995; and in revised form, January 17, 1996)
From the
Bovine, rat, and chicken UDP-GlcA:chondroitin
glucuronyltransferase activities in sera during prenatal and postnatal
development were systematically measured with polymeric chondroitin as
an exogenous acceptor and with UDP-[C]GlcA as a
donor. The results indicated that the activity changed markedly with
development in all species examined. Specifically, the activity was the
highest at the middle prenatal stage in the bovine and chicken sera and
at the late prenatal stage in the rat serum, and it decreased sharply
thereafter in all three species. Although the origin of the serum
enzyme has not yet been determined, these changes may reflect
developmentally regulated biosynthesis of chondroitin sulfate and also
suggest that the glucuronyltransferase could be a regulatory enzyme
controlling the expression of chondroitin sulfate.
A wide variety of proteoglycans with characteristic sulfated
glycosaminoglycan (GAG) ()chains exhibit tissue-specific and
developmentally regulated expression (1) and have been
implicated in the regulation and maintenance of cell proliferation,
cytodifferentiation, and tissue morphogenesis(2) . The
structures of cartilage chondroitin sulfate GAGs change with normal
embryonic development and growth or
aging(3, 4, 5) . More recently, chondroitin
sulfate has been implicated in the development of the rat central
nervous system(6, 7) . The basis for the
developmentally regulated and tissue-specific synthesis of chondroitin
sulfate and other GAGs is at present poorly understood. As the GAG
structures are largely determined by the specificities of the
glycosyltransferases and sulfotransferases responsible for their
synthesis, it is presumed that differential expression of these enzymes
is most likely the key for the controlled synthesis of GAGs. However,
few studies have systematically investigated the degree to which
specific glycosyltransferases are differentially expressed.
Chondroitin sulfate is synthesized as a proteoglycan and contains a number of chondroitin sulfate side chains covalently linked to the core protein. Chondroitin sulfate has a linear polymer structure that possesses repetitive, sulfated disaccharide units containing glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc)(8, 9) . The GlcA-GalNAc disaccharide units are synthesized by the alternating actions of glucuronyl- and N-acetylgalactosaminyltransferases(8) .
While searching for glycosyltransferases involved in chondroitin sulfate biosynthesis in various animal tissues, we detected in animal sera the two enzymes UDP-GalNAc:chondroitin N-acetylgalactosaminyltransferase (GalNAc-T) (10) and UDP-GlcA:chondroitin glucuronyltransferase (GlcA-T), which are probably responsible for polymerization of the chondroitin sulfate backbone, and found the development-dependent activity of the latter enzyme. In this report, we present evidence for developmental changes in GlcA-T activity in sera from various animal species.
Figure 1: Developmental changes in GlcA transferase and GalNAc transferase activities in bovine sera. GlcA transferase (A) and GalNAc transferase (B) activities were determined as described under ``Experimental Procedures'' and in the previous report(10) , respectively. Commercial preparations were used for the sera. Each value represents the mean ± S.E. of 4-6 specimens.
Figure 2:
Characterization of the GlcA transferase
reaction products using -glucuronidase or chondroitinase AC-II.
C-Labeled reaction products recovered from a Sephadex G-25
column were subjected to digestion with
-glucuronidase or
chondroitinase AC-II as described under ``Experimental
Procedures.'' The
-glucuronidase digest (
),
chondroitinase AC-II digest (
), or the undigested sample (
)
was applied to a column of Sephadex G-25 (1.0
105 cm), and the
respective effluent fractions were analyzed for
radioactivity.
Figure 3:
HPLC analysis of the chondroitinase AC-II
digest of the GlcA transferase reaction products. The GlcA transferase
reaction products were digested with chondroitinase AC-II, and the
resultant radioactive materials were isolated as described under
``Experimental Procedures.'' The isolated materials (5500
dpm) were analyzed by HPLC on an amine-bound silica column as described
previously(13, 14) . The eluate was collected at 30-s
intervals for radioactivity measurement by liquid scintillation
counting. Arrows indicate the elution positions of the
standard disaccharides: 1, GlcA1-3GalNAc; 2,
GlcA
1-3GalNAc; 3,
GlcA
1-3GalNAc(6-O-sulfate); 4,
GlcA
1-3GalNAc(4-O-sulfate); 5,
GlcA(2-O-sulfate)
1-3GalNAc(6-O-sulfate); 6,
GlcA
1-3GalNAc(4,6-O-disulfate).
Figure 4: Developmental changes in GlcA transferase activity in rat, bovine, chick, and human sera. The enzyme activity was determined for sera prepared from individual animals and their fetuses as described under ``Experimental Procedures.''
In this study, development-associated changes in serum GlcA-T
activity involving chondroitin sulfate synthesis were demonstrated in
various animal species, while the associated GalNAc-T activity did not
change markedly during development. These soluble enzymes presumably
represent truncated forms of the membrane-bound Golgi enzymes lacking
the transmembrane domain, since the biosynthesis of chondroitin sulfate
occurs in the Golgi apparatus where such biosynthetic enzymes normally
reside. Although it is as yet unknown from which tissues these enzymes
are secreted into the bloodstream and what their physiological
significance is, it is conceivable that the changes reflect
development-associated biosynthesis of chondroitin sulfate in the
tissues from which they originate. It has been reported in this regard
that the structures of the proteoglycan subunits in calf and human
articular cartilage change considerably between the fetal and the
mature adult stages, and especially chondroitin sulfate chains become
shorter with age(3, 4) . In addition, we have found
that cultured chick embryo chondrocytes secrete the GlcA-T into the
culture media. ()Considering the present results that the
GlcA-T activity changes with development, these observations suggest
that, although the significance of these changes in chondroitin sulfate
structure remains to be established, serum GlcA-T may originate largely
from cartilage. Highly vascularized immature cartilage containing
proliferating and differentiating cells may secrete more GlcA-T into
the vascular system than avascular adult cartilage composed of
quiescent mature chondrocytes. The results also suggest that the GlcA-T
but not the GalNAc-T could be a regulatory enzyme controlling the chain
length of chondroitin sulfate. It should be noted that the serum levels
of chondroitin 6-sulfotransferase and keratan sulfate galactose
6-sulfotransferase have also been demonstrated to change
developmentally, being markedly higher in the prenatal than postnatal
stage(15) . It is intriguing to investigate whether these
enzymes and the GlcA-T originate from the same tissue and whether their
synthesis and/or secretion are controlled in a coupled manner at the
transcriptional and/or translational level.
The differential changing patterns observed for the GlcA-T and the GalNAc-T activities (see Fig. 1) are of particular interest. The differences may indicate that these two enzymes are totally different and the mechanism of polymerization of chondroitin sulfate chains is distinct from that of heparin/heparan sulfate chains, which involves a single enzyme, GlcA/GlcNAc transferase(16) . Alternatively, the observed decrease in the GlcA-T activity during development may be associated with increasing concentrations of an unidentified inhibitor for the enzyme. Conclusive proof that the GlcA-T and the GalNAc-T are two separate enzymes will require purification and characterization of the corresponding enzymes.
The carbohydrate moieties of glycoconjugates
on the surfaces of cells are known to undergo various changes during
the malignant transformation of cells. Many of these carbohydrate
structures are described as oncofetal antigens because they are most
abundant in early fetal development, and their expression is
developmentally regulated (17, 18) . Moreover, the
antigens and sometimes the glycosyltransferases responsible for their
synthesis are actively shed into the blood from cancer cells and
therefore can be detected and measured in cancer patients' sera
as tumor markers(17) . In view of the observation that the
GlcA-T is secreted into the bloodstream in a development-associated
manner, evaluation of GlcA-T as a new tumor marker and screening tests
for a possible tumor-related increase in the GlcA-T activity in sera
from cancer patients would be of interest. In fact, appreciable enzyme
activity was observed in newborn human sera whereas little activity was
detected in normal adult sera (see Fig. 4). Furthermore, in
preliminary experiments some human cancer cell lines including a
melanoma cell line G361 (ATCC CRL-1424), an amelanotic melanoma cell
line C32 (ATCC CRL-1585), and a colon adenocarcinoma cell line LoVo
(ATCC CCL-229) were found to abundantly secrete the GlcA-T into their
culture media, suggesting that the above may indeed be the
case.