Light-induced 3-O-Sulfotransferase Expression Alters Pineal Heparan Sulfate Fine Structure

Proteoglycans are dominant glycoconjugates located on the cell surface and in extracellular spaces and consist of a core protein with one or more glycosaminoglycan side chains linked covalently. Heparan sulfate (HS) belongs to the family of glycosaminoglycans. HS has been assigned a variety of physiological and pathological functions, such as cell-cell adhesion, cell-matrix adhesion, cell proliferation, motility and differentiation, lipoprotein metabolism, blood coagulation, inflammation, tissue regeneration, tumor progression and invasion, pathogenic infection by bacteria, protozoa, and viruses, through specific interaction with a wide array of proteins, ligands, receptors, and pathogens (Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729–777). We have shown here for the first time that light induces changes in pineal HS fine structure and that occurrence of the rare 3-O sulfation catalyzed by HS 3-O-sulfotransferase (3-OST2) is predominantly restricted to daytime pineal glands.

HS 1 is ubiquitously expressed on the cell surface and has been shown to regulate many different biological functions (1). It is a highly acidic polysaccharide with repeating disaccharide units consisting of a glucosamine and hexuronic acid (idoand/or gluco-). HS is biosynthesized in the Golgi by the addition of nucleotide sugars to the reducing end of the growing polysaccharide chain followed by subsequent modification by different enzymes in a concerted fashion (2). The nascent chain may be epimerized at the C-5 position and/or sulfated at the C-2 position of uronic acid residues, and may be N-or O-sulfated and/or N-acetylated glucosamine residues. Although core proteins have fairly homogeneous compositions, the lengths and compositions of HS chains are highly variable (2). Heparan sulfate glucosaminyl 3-O-sulfotransferase (3-OST) was the first biosynthetic enzyme found to be present in multiple isoforms and carry out precursor structure specific rare 3-O modification (3). HS glucosaminyl 3-OST1 isoform was demonstrated to be important for the anticoagulant function of HS in which antithrombin III specifically binds to 3-OST1 modified HS structure, whereas 3-OST3a isoform is critical for entry of herpes simplex virus into the cell where the interaction of viral gD protein with 3-OST3a-modified HS occurs (4 -6). 3-OST2 and 3-OST4 isoforms were found to be exclusively and abundantly expressed in many different areas of the brain. However, knowledge of their functions remains elusive.
The pineal gland is a neuroendocrine organ of the brain that synthesizes and secretes melatonin, a nocturnal hormone implicated in the reproductive functions of seasonal animals, clock resetting, and sleep modulation. By differential analysis of pineal day and night mRNA, we have identified a large amount of 3-OST2 in the pineal gland in which expression is restricted to daytime (7). However, it was not known whether the increase in 3-OST2 mRNA levels would lead to an altered HS fine structure. On the one hand, immunological reagents to specifically detect 3-OST2 are not available. On the other hand, the presence of an appropriate HS precursor structure in pineal glands for modification by 3-OST2 is unknown. For these reasons, we investigated whether light induces changes in pineal HS fine structure because of a rare 3-O modification. This may provide novel molecular insights into the link between heparan sulfate glycobiology and the largely elusive circadian biology.

EXPERIMENTAL PROCEDURES
Materials-Heparitinase I, II, and III derived from Flavobacterium heparinium were purchased from Seikagaku USA (Cape Cod, MA). All chemicals and LC/MS grade solvents were from either Sigma or Aldrich. DEAE-Sephacel material was purchased from Amersham Biosciences. Adult male Sprague-Dawley rats purchased from Harlan Sprague-Dawley were housed in a temperature-controlled room under 14:10 light/dark conditions with lights-off at 1 a.m. for more than 1 week before experiments. Day pineals were harvested at 2 p.m., and the night pineals were isolated at 7 a.m. under red light and immediately placed in dry ice.
Isolation of Pineal Heparan Sulfate-Pineals were homogenized and treated with Pronase solution (150 g of protease type XIV from Streptomyces griseus in 1 ml of 40 mM NaOAc, 320 mM NaCl, pH 6.5). Proteolysis was carried out at 37°C for 24 h. The digestion mixture was then purified as described earlier to isolate heparan sulfate for subsequent LC/MS characterization (8).
Enzymatic Digestion of Day and Night Pineal Heparan Sulfate Polysaccharides-HS isolated from five day and five night pineals was digested with 0.33 milliunits of heparitinase I, II, and III in 50 l of 40 mM ammonium acetate buffer (pH 7.0) containing 3.3 mM CaCl 2 at 22°C for 24 h. Then the reaction mixture was taken out and boiled at 100°C for 2 min to inactivate the enzyme, and a fraction of reaction mixture (10 l) was directly loaded to capillary HPLC for LC/MS analysis as we described previously (8).

RESULTS AND DISCUSSION
Previously we demonstrated that 3-OST2 expression is abundant during the daytime and is light-inducible at night through the suppression of ␤-adrenergic signaling (7). Despite this finding, it was unclear whether induced 3-OST2 RNA * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. expression is associated with increased 3-OST2 enzymatic activity and the generation of a novel HS structure within the pineal gland. In this communication, we demonstrate that light-induced expression of 3-OST2 leads to the generation of a novel 3-OST2-modified HS as determined by MS ( Fig. 1 and Table I). Our ability to detect this specie was dependent upon the use of the newly developed capillary HPLC coupled to electrospray ionization-time-of-flight MS (8). This approach is very sensitive and can detect minute quantities of HS. The differences at the disaccharide level were obtained by enzymatic degradation of day and night pineal derived HS. The difference in the amount of tetrasulfated disaccharide is greater than the differences in the amount of any other disaccharides as a result of differences in 3-OST2 level regulated by light. Furthermore, this specific tetrasulfated disaccharide contains a 3-O-sulfated glucosamine unit, which is the result of 3-OST2 action. We have shown previously that 3-OST2-modified glucosamine units located to reducing side of 2-O-sulfated uronic acid, whereas 3-OST1 isoform fails to modify glucosamine units located to the reducing side of 2-O-sulfated uronic acids (9). We have found that these disaccharides constitute the molecular signature of the action of 3-OST2 enzymes. 2 The disaccharides were resolved to homogeneity by capillary HPLC and were then analyzed by on-line coupled electrospray ionization mass spectrometry. The observed m/z of 785 corresponds to a tetrasulfated disaccharide molecular ion ([M Ϫ 2H ϩ 1DBA] Ϫ1 , Fig. 1 (Day  Pineal), and thus the structure is ⌬ U2S-GlcNS3S6S. The mass spectrum clearly shows the differences in the 3-O-sulfated disaccharides derived from the digestion of night and day pineal HS polysaccharides. This difference corroborates the nature of the modification controlled by day and night cycle. It seems probable that this light-induced modified HS interacts with a specific protein within or outside of the pineal and may be a part of the molecular machinery that is responsible for circadian rhythm. We are currently attempting to identify this putative downstream target by a combination of proteomic and synthetic heparanomic approaches (10)(11)(12). This is likely to represent a novel pathway not involving melatonin as suggested by Borjigin et al. (7). We are also currently attempting to understand the mechanism by which light influences the transcription of the pineal 3-OST2. As light acts indirectly by termination of the adrenergic signaling to the pineal gland, and ␤-adrenergic (7) and cAMP signaling 3 suppress the 3-OST2 transcription, we hypothesize that a cAMP response element is present in the 3-OST2 promoter and that the transcriptional activation of the 3-OST2 gene requires the silencing of the cAMP response element. We have previously shown that 3-OST2 enzyme preferentially sulfates glucosamine residues that are located to the reducing side of 2-O-sulfated glucuronic acid, which is known to be present in various areas of the brain (13). In the present study, we utilized heparin lyases to cleave the minute quantity of pineal HS into disaccharides for the structural analysis, although with the loss of stereochemical information of uronic acids. In order to determine unequivocally the structures in the vicinity of the 3-Osulfated glucosamine residues, it is essential to utilize other cleavage techniques that are unfortunately not compatible with mass spectrometry at the present time. We surmise that the pineal HS fine structure contains these two unusual residues that may specifically interact with a protein ligand to regulate a hitherto unknown pathway involved in circadian biology. In summary, this communication for the first time demonstrates the involvement of an HS fine structure in circadian rhythm. It is likely that other neurobiological functions may also be subserved by this brainspecific sulfotransferase (3,4).