Showing papers by "Ajit Varki published in 1996"

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Abstract: Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues Thus the selectins have generated much interest as targets for antiinflammatory agents Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics

Calcium-dependent oligonucleotide antagonists specific for L-selectin.

Abstract: The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

The times they are still a'changing: keeping up with the times.

Abstract: The woods are lovely, dark and deep. But I have promises to keep, And miles to go before I sleep. Robert Frost Following a 70-year-old tradition, the JCI will change editorial command again in 1997. The search for a new editor is underway, and applications from many excellent candidates are under review. Most observers agree that the tenure of the current editorial group has been associated with improvements in the image and functioning of The journal. In times past, it would have been reasonable for the incumbents to rest on their laurels and coast to the finish without further ado. However, these are not ordinary times. As Toffler predicted (1), the rate of change has continued to accelerate , and a year is now too long a time to stay still. What follows is an interim report and an agenda for 1996 that is influenced by the need to keep The journal at the cutting edge. Improvements in the review process. Introduction of the uniform manuscript submission form and of manuscript revision checklists have streamlined submissions and handling and reduced procedural delays. The review process has been overhauled and improved. Computerization of manuscript tracking and liberal use of overnight mail services and electronic communication have reduced the median time from submission to first decision to a little over 30 days. Some further improvements can be expected with electronic handling of manuscripts and reviews. Evaluation of the novel screening review system introduced in 1992 indicates that it is efficient and does not unduly compromise fairness and objectivity (2). While no peer review system is perfect, this screening approach appears, in balance, to be best for all concerned (authors , reviewers, and editors). If imitation is indeed the sincerest form of flattery, the Editors are pleased to note many obvious examples wherein other journals have copied the forms, procedures, and review policies recently developed by the JCI. Increasing volume of manuscripts received. Despite the appearance of many new journals that are potential competitors, submissions continue to rise, having increased by almost 50% in the last five years and by almost 12% in 1995. As a nonprofit publication with limited space, the JCI now has an acceptance rate of ‫ف‬ 20%, with projected rates for the future being lower. While this makes Editorial decisions ever more difficult, it ensures that The Journal need publish nothing but the best selections from among its submissions. …

Does DG42 synthesize hyaluronan or chitin?: A controversy about oligosaccharides in vertebrate development.

Abstract: Science generally progresses in slow but deliberate increments, which are punctuated by major advances in concept or fact. However, the latter are rare and not infrequently go unrecognized when they first occur. Another event that can add spice to a field, and attract the attention of scientists from outside the discipline, is a genuine controversy. One such controversy is presented by two reports in this issue of the Proceedings (1, 2). To be asked to referee such a controversy is an interesting but difficult task, since both groups have significant data to back their claims. The story begins in 1983, when Igor Dawid and colleagues (1) reported the isolation of several genes that are Differentially expressed at Gastrulation (DG) in embryos of the frog, Xenopus laevis (3). One of these, the endoderm-specific DG42, is expressed in a short window during embryogenesis, being first detected after the midblastula stage, peaking at late gastrula, and decaying by the end of neuralation (4-6). Appropriate probes were used to show that the messenger RNA and predicted protein product move in a wave or gradient through the embryo, with the last remnants seen in the ventral regions of the gut at the tailbud stage. For a while thereafter, DG42 remained an interesting gene in search of a function. As often happens, the first clues came from unexpected sequence homology information. When it was first cloned, DG42 showed no obvious homologies to any previously known protein or gene. Subsequently, some similarities were found with fungal chitin synthases (7) and with the rhizobium NodC gene that is known to synthesize chitin oligomers (8-10). What is chitin? It is a repeating ,B1-4-linked homopolymer of the monosaccharide GlcNAc (see Fig. 1) that is one of the most widespread and abundant molecules in the biosphere, providing, for example, a major component of the cell walls of fungi and the shells of crustaceans and arthropods (11, 12). This important structural role for the extended polysaccharide may seem of little relevance to vertebrate development. However, shorter oligomers of the same repeating sequence are known to be soluble "oligosaccharins," mediating short range hormonal responses between Rhizobium bacteria and leguminous plants during the process of nitrogen-fixing root-nodule formation (13-15). Indeed, complex structural variations on the theme of the basic chitin backbone are well known to mediate a variety of specific interactions between bacteria and plants (for some examples, see refs. 16-20). Intrigued by these homologies, Semino and Robbins (21) then showed that when generated in an in vitro transcription/ translation system, the DG42 gene product was capable of synthesizing both short chitin oligomers and some larger products. The required sugar nucleotide donor was UDPGlcNAc; the products had the correct chromatographic properties, and they were degraded appropriately by a bacterial chitinase. Thus, DG42 was proposed to be the first recognized vertebrate chitooligosaccharide synthase (21). However, another interesting homology had also appeared between DG42 and the hasA gene of Streptococci (22, 23). The latter is responsible for the synthesis of another repeating polymer of sugars called hyaluronan. What is hyaluronan? It is a polymer consisting of alternating units of ,B1-4-linked GlcNAc and ,B1-3-linked glucuronic acid (GlcA, see Fig. 1). At first glance, these may seem to be very similar structures. Indeed, the linkages are very similar, and the donor nucleotides for both units are based on UDP (UDP-GlcNAc and UDP-GlcA). However, the similarity ends there (24, 25). Partly by virtue of its carboxylate groups, hyaluronan has physical properties that are almost diametrically opposite to those of chitin, being capable of retaining large amounts of water to form a gel. Furthermore, unlike chitin, hyaluronan expression is primarily reported in vertebrates, and in a few pathogenic bacteria such as group A and C Streptococci (24, 25). In view of these homologies, Semino and Robbins had also checked to see if the DG42 protein had hyaluronan synthase activity in vitro, but did not find any (21). This seemed to settle the issue that the DG42 gene product was primarily a chitin synthase. Enter the new study of Meyer and Kreil (1), which shows that rabbit kidney and human osteosarcoma cells induced to express the DG42 gene with a vaccinia virus system synthesize increased amounts of hyaluronan. Lysates and membranes from such transfected cells showed markedly increased hyaluronan synthase activity, which required the addition of both UDP-GlcNAc and UDP-GlcA donors. The product of the reaction was sensitive to hyaluronidases, but not to chitinases, and appropriate controls showed that the overexpression of hyaluronan synthesis was clearly related to DG42 expression (1). These authors conclude that their results are at variance with the earlier report of Semino and Robbins (21). Meanwhile, the latter group have an update to their story that is also published in this issue (2). They now show that DG42 homologues and their protein products are expressed in early embryos of zebrafish and mouse during the gastrula-early neuralation stages, and that chitin-oligosaccharide synthesis can be detected in extracts from these sources as well. Furthermore, this activity was immunoprecipitated by a DG42specific antibody (4) provided by Dawid. Also, overexpression of DG42 in a different cell type (mouse 3T3 cells) gives the synthesis of chitooligosaccharides, but no increase in background levels of hyaluronan synthesis. Finally, these authors show a physical separation of chitin synthase activity from most (but not all) of the hyaluronan synthase activity in embryo extracts (2). How can one reconcile the findings of the two studies and determine the true role of DG42? Semino et al. do make one preliminary attempt to do so (2). They state that commercial preparations of hyaluronan have chitin oligomers at their reducing end core region (further details are evidently to be published elsewhere). They suggest that DG42 might function to produce chitin oligomers that act as templates for hyaluronan synthesis (see Fig. 1). In this regard, it is interesting that Meyer and Kreil note a requirement for high concentrations of UDP-GlcNAc in their reactions (1). To consider this possibility further, let us review what is known about hyaluronan synthesis in vertebrate systems. The biosynthesis of this polysaccharide is peculiar, in that it follows a route different from that taken by most other molecules

Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding

Abstract: Previous studies have shown that the mucin-type polypeptides GlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectin only when they are synthesized by the specialized high-endothelial venules (HEV) of lymph modes. Since sialylation, sulfation, and possibly fucosylation are required for generating recognition, we reasoned that other mucins known to have such components might also bind L-selectin. We show here that soluble mucins secreted by human colon carcinoma cells, as well as those derived from human bronchial mucus can bind to human L-selectin in a calcium-dependent manner. As with Gly-CAM-1 synthesized by lymph node HEV, alpha 2-3 linked sialic acids and sulfation seem to play a critical role in generating this L-selectin binding. In each case, only a subset of the mucin molecules is recognized by L-selectin. Binding is not destroyed by boiling, suggesting that recognition may be based primarily upon carbohydrate structures. Despite this, O-linked oligosaccharide chains released from these ligands by beta-elimination do not show any detectable binding to L-selectin. Following protease treatment of the ligands, binding persists in a subset of the resulting fragments, indicating that specific recognition is determined by certain regions of the original mucins. However, O-linked oligosaccharides released from the subset of non-binding mucin fragments do not show very different size and charge profiles compared to those that do bind. Furthermore, studies with polylactosamine-degrading endoglycosidases suggest that the core structures involved in generating binding can vary among the different ligands. Taken together, these data indicate that a single unique oligosaccharide structure may not be responsible for high-affinity binding. Rather, diverse mucins with sialylated, sulfated, fucosylated lactosamine-type O-linked oligosaccharides can generate high-affinity L-selectin ligands, but only when they present these chains in unique spacing and/or clustered combinations, presumably dictated by the polypeptide backbone.

“Unusual” modifications and variations of vertebrate oligosaccharides: are we missing the flowers for the trees?

Abstract: The remarkable complexity of oligosaccharide structures found on vertebrate cells results from the concerted action of glycosyltransferase enzymes (Joziasse, 1992; Stanley and Ioffe, 1995; Varki and Marth, 1995; Whitfield and Douglas, 1996), several of which were first identified and characterized by Robert Hill (Paulson et al, 1977; Paulson et al, 1978; Beyer et al, 1979; Rearick et al, 1979; Sadler et ai, 1979, 1982). Together with the structural characterization of naturally occurring oligosaccharides, the study of these enzymes has led to the definition of the major structural motifs of vertebrate sugar chains (Cummings, 1992; Kobata and Takasaki, 1992; Varki and Freeze, 1994; Varki and Marth, 1995). These motifs can be divided into (1) the core regions unique for each class of glycoconjugate (e.g., the Chitobiosyl-N-Asn-linkage region); and (2) the outer chains (e.g., type 1 and 2 lactosamine units with sialylation and/or fucosylation) that can be shared to varying extents by the different core classes (Cummings, 1992; Kobata and Takasaki, 1992; Varki and Freeze, 1994; Varki and Marth, 1995). Vertebrate cells utilize a relatively limited subset of the monosaccharides known to exist in nature, and in only some of the many possible combinations. However, several unusual variations of the typical structures exist, as well as a variety of specific modifications of the individual monosaccharide units. Hypothesizing that such subtle variations and modifications are more likely to mediate specific biological functions (Varki, 1993), we have paid special attention to their existence, structure and biosynthesis. Here I briefly describe some examples that my group has studied over the past decade.

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

Abstract: The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.