Search

The biogeographic history of aquatic organisms in relation to the Wasatch Mountains divide (which separates the eastern Great Basin and upper Colorado River basin in Utah) has been little studied aside from a large body of work on fishes. Pyrgulopsis kolobensis is a small springsnail that is distributed (in the eastern portion of its range) along the western flanks of the Wasatch Mountains, with a single population occurring just across the Wasatch divide in Strawberry Valley. Here we analyze the genetic structure of this species across the Wasatch divide (using the mtCOI gene) to discriminate between alternative hypotheses that explain this distributional pattern. The 6 P. kolobensis populations that we sampled were resolved as a single, weakly supported and shallowly structured clade in a Bayesian analysis. Specimens from Strawberry Valley shared a unique haplotype and differed from the other populations by 0.3%–0.8% sequence divergence, suggesting a geologically recent split that well postdat...

Placobdella sophieae Oceguera-Figueroa et al., 2010 (Hirudinida: Glossiphoniidae) is reported from Oregon, California, and British Columbia for the first time. New hosts reported for P. sophieae include Taricha granulosa (rough- skinned newt), Rana pretiosa (Oregon spotted frog), and Anaxyrus boreas (western toad). Placobdella sophieae exhibits relatively low host specificity and all amphibians occurring in the Pacific Northwest are potential hosts.

I determined the distribution of limpets (Gastropoda: Ancylidae: Ferrissia) in the Colorado River and Rio Grande basins by handpicking specimens from the undersides of rocks and vegetation at 16 sites from a total of 495 surveyed sites in Colorado, Utah, New Mexico, and Arizona. Shell morphology, including morphometries, was compared to species holotype descriptions and museum lots. Ferrissia rivularis occurred in the upper Colorado, Gunnison, and San Juan rivers, and in the upper Rio Grande basin. Ferrissia walkeri, hereafter called the Walker morph, occurred only in the Gila River basin of Arizona and New Mexico. I reviewed the problems of classification of the Walker morph as being either F. walkeri, described from Arkansas and noted in Michigan and Baja California Sur, or Ferrissia fragilis, described from California and noted from the eastern United States. The Walker morph is identified by its vertical trending protoconch, which lies well within the right posterior quadrant, with the apex ...

Leech species of the mid-Snake River of Idaho and Oregon are described, and the distribution of the extant leech Mooreobdella microstoma Moore in the Snake River paleodrainage is delineated. Samples were collected from aquatic surveys in the Snake River using suction dredging by the Idaho Power Company and U.S. Bureau of Reclamation between 1995 and 2006. Supplementing these surveys, opportunities were provided for leech identification in water-quality analyses in Arizona and Wyoming and in other surveys in California, Oregon, Washington, and Idaho. Eight species of leeches were found in the Snake River surveys. Erpobdella parva Moore was the most widely distributed species, occurring both above and below Shoshone Falls. Mooreobdella microstoma was widely distributed below Shoshone Falls. Other leech species were rare, although Helobdella stagnalis Linnaeus was very common above and less common below Shoshone Falls, a natural barrier to the anadromous fish. Mooreobdella microstoma is an extant sp...

Field collections at more than 2900 sites and the examination of many museum collections and literature allowed me to map the historical and current distribution of several freshwater molluscan faunal groups in the Intermountain region of the United States (Great Basin, Colorado River drainage basin, and upper Snake River sub-basin). Historical and current records show that Margaritifera falcata, Anodonta californiensis, and Ferrissia rivularis have drainage-specific distributions, while Valvata utahensis has a specific drainage pattern, and V. californica (new combination) has a dispersed pattern. Shell morphometric data of Valvata and Ferrissia show extensive shell variation between and within populations. Current surveys show that these molluscan populations have been reduced since the colonization by European descendants over the last 150 years. Margaritifera falcata was found to be extirpated from eastern California, Nevada, and Utah and was common in only 1 stream. Anodonta californiensis p...

Prior analyses of recombinant CD44 fusion proteins have indicated that combinatorial splicing of variant exons exerts distal effects on chondroitin sulfate content and structure, which may regulate the biological properties of the respective CD44 isoforms. The consequences of splicing of variant exons V4-7 on the heparan sulfate moieties were therefore examined, utilizing recombinant chimeras containing exons V3 and V8-10, engineered with or without exons V4-7 and expressed as Ig fusion proteins in COS cells. Splicing of exons V4-7, though they contain no consensus motifs for glycosaminoglycan assembly, resulted in markedly increased polymer sulfation levels of the heparan sulfates. The sulfate groups of both the CD44 V3-10 and V3,8-10 isoforms occurred as di- and tri-sulfated dissacharide units and were restricted to one N-sulfated block domain within the polymers. Compared to native human keratinocyte CD44, the recombinant heparan sulfates were relatively low in sulfate content. Our data indicate that variant exon V4-7 splicing exerts distal effects on the composition of this glycosaminoglycan. These effects may regulate those functions that are mediated through the heparan sulfate moieties, such as the binding of growth factors.

The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura ). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given.

Previous studies have indicated that CD44 isoforms, spliced with variant exons, are heterogeneously glycanated with chondroitin sulphate and heparan sulphate chains. Because such alternative splicing may regulate divergent biological effects of the specific isoforms, we analysed the consequences of this process on the composition and structure of the chondroitin-sulphate chains. Recombinant chimaeras were engineered with and without exons V3-10 or V3,8-10 and expressed as Ig fusion proteins in COS cells. In addition, the chondroitin sulphates of wild-type isoforms were contrasted with those of isoforms mutated with serine-to-alanine codon substitutions at a putative Ser-Gly-Ser-Gly glycosaminoglycan acceptor site within exon V3. The chondroitin sulphates contained both 4- and 6-sulphated galactosamine residues, although there was a high content of non-sulphated galactosamine-containing repeat units. Splicing of exons V4-7, which contain no Ser-Gly consensus motifs, resulted in increased glycanation with chondroitin-sulphate chains, as well as increased sulphation levels of the polymers. Comparison of wild-type and acceptor-site mutant isoforms showed that chondroitin-sulphate content declined by more than 60-80% in the mutant, indicating that assembly of chondroitin-sulphate chains occurs there, and a general decrease in the sulphation level of the remaining chains was observed. Undersulphation of the recombinant chondroitin sulphates was shown by parallel analyses with native human keratinocyte CD44 molecules and is most probably an artifact of transient expression in COS cells. Our data indicate that combinatorial exon splicing exerts complex and distal effects on glycanation patterns and structure, which presumably modulate those functions that may be mediated though the chondroitin-sulphate moieties, such as motility and matrix invasion.

The artesian springs of Tule Valley are similar to those of adjacent Snake Valley and Fish Springs Flat based on conductivity and temperature. All three valleys support Ranidae amphibians and the leechErpobdella punctata. The artesian springs in Snake Valley and Fish Springs Flat contain six and two species of fish and contained up to 18 and 12 species of mollusk respectively, whereas Tule Valley artesian springs contain neither fish nor mollusks. The leechesHelobdella stagnalis, Glossiphonia complanata, andHaemopis grandis were found in Snake Valley whereasHelobdella triserialis, Theromyzon rude, andHaemopis marmorata were found in Tule Valley. These springs which were covered by Lake Bonneville to a depth of several hundred meters, 16 000 BP., became isolated after the paleolake desiccated 13 000 years BP. The marsh snailCatinella is found above the paleolake level in Snake and Tule Valley and has not penetrated to the valley floor habitats once covered by the paleolake, whereas another marsh snailOxyloma has penetrated into these habitats in Snake Valley. The leech and molluscan distributions in Tule, Snake and Fish Springs Valleys suggest that the paleolake did not allow for much movement among the valleys, and successful passive aerial transport has not occurred after the paleolake desiccation 13 000 years BP. Paleozoological models are proposed to explain the presence and absence of these species in Tule Valley. Both lateral movement (along paleolake shorelines) and vertical movement (to new habitats formed after the desiccation of the paleolake) by amphibians, mollusks and leeches is restricted in large terminal lakes and is species dependent in both spatial and temporal scales of the hydrological cycle.

Cultured fibroblasts, bovine aortic endothelial cells, and human keratinocytes synthesize both proteoglycans and glycosaminoglycan free chains, the proportions varying between cell types. The major metabolic labeling is in proteoglycans, except for keratinocytes with approximately 60% of product as free chains. The proteoglycans range from approximately 50- greater than 1000 kDa, and the glycosaminoglycan side chains derived by alkaline elimination are approximately 30- greater than 100 kDa. The glycosaminoglycan free chains, in contrast, are smaller, from approximately 7-40 kDa in mass. The proteoglycans are both medium and cell layer constituents, whereas the glycosaminoglycan free chains are essentially confined to cells. The cellular proteoglycans and a portion of the free chains are accessible to in situ digestion by Flavobacterial glycosaminoglycan lyases, presumably reflecting localization to the cell surface. Collectively, the data show the free chains to be a common feature of all cells studied and to be partly expressed on cell surfaces. We hypothesize that the processing that creates these free chains occurs on cell surfaces, in which location they could serve ligand receptor functions.

The freshwater leeches (Hirudinida) in the Province of Newfoundland and Labrador were investigated by examining theliterature, the Canadian Museum of Nature and the United States National Museum of Natural History records, and aleech survey. New pigmentation forms are described for Erpobdella punctata (Leidy) and Erpobdella obscura (Verrill).This is the first published record for Haemopis lateromaculata Mathers in Canada. Four species (Glossiphonia elegans(Verrill), Helobdella modesta (Verrill), Erpobdella punctata, and Erpobdella obscura) were found in both Labrador andNewfoundland with Erpobdella obscura common in Labrador and the other three species common in Newfoundland.Seven other species of leeches were less abundant in Newfoundland with 6 of these species very restricted in distribution.The abundance of leech species in Newfoundland and the paucity of leech species in Labrador suggested that theisland species were present in a Pleistocene refugium associated with Newfoundland or the Grand Banks. Post-Pleistocenebarriers to leech mobility are examined, and possible timing of colonization events is proposed in this model.

The leech hyaluronoglucuronidase (hyaluronidase I) was identified in Erpobdellidae (Nephelopsis obscura and Erpobdella punctata) and Glossiphoniidae (Desserobdella picta) and historically described from Hirudinidae (Hirudo medicinalis). A second leech hyaluronidase (hyaluronidase II) which hydrolyzed only a few bonds to for hyaluronan oligosaccharides larger than 6500 Da, was found in Glossiphoniidae (Helobdella stagnalis, Glossiphonia complanata, Placobdella ornata, and Theromyzon sp.) and in Haemopidae (Haemopis marmorata). The distribution of the two hyaluronidases in leech occurred in both orders (Arhynchobdellida and Rhynchobdellida) and in macrophagous and haematophagous feeding types whereas the liquidosomatophagous leeches only had hyaluronidase II.

Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [35S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.

The synthesis of glycosaminoglycans (GAG) in a freshwater mussel was studied in organ culture using labeled precursors. The major GAGs synthesized were determined and characterized by chemical and enzymatic methods. They were shown to be heparin and an unusual type of heparan sulfate. Gills produced about 50% of each polymer; mantles synthesized little heparin and mostly the heparan-sulfate-like compound, which is similar to a GAG isolated previously from lobsters. No significant amounts of chondroitin sulfates were present. Histological data showed that the sulfate-labeled GAGs were present mainly in exterior pericellular and basement membrane locations of gills and mantle. That is, they would be in contact with the external aqueous environment, suggesting a potential role in calcium transport and storage.

The metabolic relationships between heparan sulfate proteoglycans, free chains, and oligosaccharides in different cell locations were evaluated by comparing their glycosaminoglycan structure. Metabolically labeled heparan sulfate proteoglycans of BALB/c 3T3 cell layers and in conditioned medium were compared with the heparan sulfate free chains (modal mass = 10 kDa) and oligosaccharides (modal mass = 3 kDa) of the cells. Nonlytic, in situ digestion with heparitinase I indicated that 90% of proteoglycans, 70% of the free chains, and 20% of the oligosaccharides were enzyme accessible, but there was no evidence using competitive iigands for binding of the products to the cell surface via the glycosaminoglycan moieties. Structurally, the membrane proteoglycans were the most O-IN-sulfated and yielded more tri- and tetra-sulfated di- and tetra-saccharides by nitrous acid degradation. In contrast, the side chanis of medium proteoglycans were less sulfated and more polydisperse in mass, suggesting that most medium proteoglycans are not processed from membrane precursors. The heparan sulfate free chains were of lower mass, less sulfated, and more heterogeneous in distribution of the anionic groups than were proteoglycan side chains. Corroborating analytical heparitinase I digestion indicted that generation of di- and tetra-saccharides proportion-ately increased from membrane proteoglycan, to cell free chain, to medium proteoglycan categories. Because the structural patterns of the heparan sulfate free chains did not reveal a clear relationship with the side chains of the major proteoglycans, their origin was further probed by [3H]BH4-labeling of the reducing terminus under varying stringencies. The end-labeled residues obtained by nitrous or strong acid hydrolysis of the free chains showed insignificant amounts of galactose and xylose, but rather glucosamine N-sulfate and a residue likely generated from glucuronate. The effective labeling that was achieved with weak alkali indicated that covalent oligopeptide is not present. In summary, the heparan sulfate free chains, which in part are components of the cell surface, are of relatively low mass, are unassociated with covalent peptide, and most probably have a disaccharide motif of glucosamine N-sulfate and a uronate residue at the reducing end. Taken together, these observations suggest that the free chains originate by processing of precursor heparan sulfate proteoglycans on the cell surface via an endoglycosidase acting on an N-sulfated portoin of the original polymer.

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.

The ability of heparan sulfate, an endogenous component of the glomerulus, to regulate the growth of cultured rat mesangial cells was investigated. Heparan sulfate caused a dose-dependent inhibition of rat mesangial cell growth, 85% inhibition compared with controls at the highest dose (1,000 micrograms/ml). Chondroitin sulfate produced no inhibition. The low-sulfated fraction of heparan sulfate (9%) produced more inhibition than the high-sulfated fraction (13%), 90 +/- 1 vs. 71 +/- 2% (P = 0.002). The effects of the heparan sulfate were completely reversible. Treatment of heparan sulfate with heparitinase increased the degree of inhibition, 71 +/- 1 vs. 84 +/- 1% (P less than 0.001). Four different oligosaccharides derived from heparan sulfate and heparin were tested for their ability to inhibit growth. One of the oligosaccharides, low-sulfated (10%), caused significant inhibition, 76 +/- 2%. Heparan sulfate was also able to inhibit the growth of Swiss 3T3 fibroblasts (63 +/- 5%). This inhibition was less marked than that seen with mesangial cells. Thus heparan sulfate was able to significantly inhibit rat mesangial cell growth in culture. Alterations in glomerular heparan sulfate may play an important role in alterations in mesangial cell growth.

The geographic range of Haemopis lateromaculata Mathers 1963 (Hirudinea: Haemopidae) is extended across North America. Its distribution in the coastal region of Alaska and British Columbia suggests a coastal Pleistocene refugia separate from the populations in the lower United States and suggests that H. lateromaculata and the Eurasian H. sanguisuga Linnaeus 1758 are sister taxa. Support of the identification and geography is based on the anatomical positions of the reproductive organs in H. lateromaculata and H. marmorata Say 1824. The variations within these species are described, noting that no specific variation was confined to a geographical region.

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.

The synthesis and turnover of metabolically labeled proteoglycans from medium, cell layer, and substratum-associated compartments were characterized in four cell lines of fibroblastic origin, including a fibrosarcoma line, and in the murine melanoma cell type, B16.F10. Substantial differences were apparent between the various cell types with regard to quantities, hydrodynamic sizes, and compartmentalization of labeled product. Such variations were greater between the different cell lines than between separately labeled cultures of the same cell type. Greater than 85% of cell-associated proteoglycans were accessible to glycosaminoglycan-degrading enzymes added to the medium of monolayer cultures, demonstrating their principal location to be external to the cell membrane. Apparent glycosaminoglycan free chains, determined by a lack of change in hydrodynamic size following alkaline elimination, were among the products from each cell line and were similarly found to be in a principally pericellular location. Results from label-chase studies demonstrated apparent independent kinetics for proteoglycans and glycosaminoglycan free chains, with little conclusive evidence for precursor-product relationships. Also, their processing by the cells was different, since the proteoglycans were shed largely unchanged into the medium for the three cell lines evaluated, whereas the free chains were not recoverable from the medium in significant amounts. The latter observation suggests the internalization of cell surface-associated free chains and their depolymerization at an intracellular site. The results, which indicate that the content, cellular disposition, and turnover of proteoglycans are quite variable between the cell lines studied, may reflect fundamental cell type-specific specialization in the metabolism of these complex substances. Furthermore, the data raise the interesting possibility that glycosaminoglycan free chains may have biological functions at the cellular level, independent of proteoglycans.

Changes in heparan sulfate correlate with increased glomerular permeability. The glomerular capillary wall functions as both a size-selective and charge-selective barrier. Heparan sulfate is known to be an important component of the charge-selective barrier to filtration of polyanions. We studied the alterations in both the charge and size selectivity barriers in a model of experimental membranous nephropathy in the rabbit. The fractional clearance of both charged and uncharged dextrans compared to inulin was measured. Sulfate incorporation into glycosaminoglycans was measured and the glomerular heparan sulfate was isolated and biochemically characterized. Membranous nephropathy in the rabbit was induced with daily injections of cationic bovine serum albumin. After three weeks of injection animals had 735 ± 196 mg/24 hours of protein excretion. There was no change in [35S] incorporation in 24 hours by experimental animals, 440 ± 91 DPM/mg dry weight of glomeruli, N = 9 versus 410 ± 98, N = 11 in controls. The percentage of [35S] incorporated into heparan sulfate versus chondroitin sulfate was decreased, 60% ± 3 versus 79% ± 2, P < 0.001. Heparan sulfate from membranous nephropathy eluted from ion exchange chromatography in a lower molarity salt, indicating a lower effective charge. Fractional clearance of neutral dextrans was significantly increased in membranous nephropathy for dextrans greater than 48 Å, while fractional clearance of dextran sulfates was significantly increased compared to controls for dextrans greater than 32 Å. Thus, in membranous nephropathy there is loss of both charge selectivity and size selectivity. The loss of charge selectivity correlated with a change in the structure of the glomerular heparan sulfate. The change in structure of the heparan sulfate could be the initiating event in the altered glomerular permeability of experimental membranous nephropathy.

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3–34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.

Three tetrasaccharides representing major structural sequences of heparin were isolated in good yield and characterized after degradation of heparin by purified flavobacterial heparinase. N -Desulfation was necessary to achieve good separation of these closely related compounds from each other. One of the tetrasaccharides was shown to be derived from the fully sulfated repeating segments; to contain l -iduronic acid and six sulfate groups, and have the structure Δ 4,5 -Hex p A-(2-SO 4 )-(1→4)-α- d -Glc p N-( N -SO 4 )-(6-SO 4 )-(1→4)-α- l -Ido p A-(2-SO 4 )-(1→4)- d -GlcN- ( N -SO 4 )-(6-SO 4 ). The second contained a d -glucuronic acid unit that was nonsulfated instead of the l -iduronic acid, and the third, obtained in a fairly low yield, contained five sulfate groups, three of which being located on the disaccharide at the nonreducing end, and having the structure Δ 4,5 -Hex p A-(2-SO 4 )-(1→4)-α- d -Glc p N-( N -SO 4 )-(6-SO 4 )-(1→4)-α- l -Ido p A-(2-SO 4 )-(1→4)- d -GlcN- ( N -SO 4 ). All tetrasaccharides had a sulfated, unsaturated uronic acid unit at the nonreducing end, confirming that the heparinase requires sulfated l -iduronic acid units for activity.

Heparan sulfate and chondroitin sulfate glycosaminoglycans of BALB/c 3T3 fibroblasts, metabolically labeled with [3H]glucosamine and [35S]sulfate precursors, are resolved by preparative Sepharose CL-4B chromatography into distinct products, the proteoglycans and the glycosaminoglycan free chains, the latter resistant to appreciable molecular weight shift upon alkaline borohydride reduction. The in situ localization of these cell layer molecules was probed with glycosaminoglycan degrading enzymes (lyases) of bacterial origin, which were used to digest isotopically prelabeled monolayer cultures prior to extraction with a nonionic detergent in the presence of protease inhibitors. Most of the total cellular complement of glycosaminoglycan free chains, in addition to the proteoglycans, proved accessible to the lyases under conditions which did not appreciably affect cell viability or morphology. Because these results were also obtained under low temperature (4 degrees C) conditions and in the presence of phenylarsine oxide, a sulfhydryl reagent that irreversibly inhibits endocytosis, the effects of the lyases are not dependent upon internalization by the cells. The cellular production and cell surface expression of the glycosaminoglycan free chains were not materially altered when lysosomal function was pharmacologically inhibited, confirming that the free chains are not intracellular intermediates in the lysosomal degradation pathways of proteoglycans. Contrary to the prevailing model, our observations establish that, at least in the cell line under study, glycosaminoglycan free chains are located on the external leaflet of the plasma membrane, as such suggesting that these products are biologically active components of cell surfaces.

A non-sulfated, N-acetylated disaccharide was isolated from hepatirin sulfate after enzymatic digestion. This compound containing α, β unsaturated uronic acid is an isomer of a disaccharide obtained previously from hyaluronic acid. It contains an α- d -(1 → 4) rather than the β- d -(1 → 3) linkage found in the hyaluronate compound. The isolation of another non-sulfated, N-acetylated compound, most likely a tetrasaccharide, indicates that at least two or more N-acetylated units occur in sequence in the polysaccharide. The linkages in the non-sulfated portion of heparitin sulfate appear to be mainly α- d -(1 → 4) .

Crude enzyme obtained from heparin-induced flavobacteria has been fractionated into a heparitinase acting on heparitin sulfates and related compounds and a heparinase acting mainly on heparin. Purification achieved for each was from 50 to 100 times that of earlier preparations containing a mixture of the two enzymes. In agreement with previous data both enzymes act as eliminases rather than hydrolases yielding products containing Δ4,5-unsaturated uronic acid. Specificity of the heparitinase appears to require the absence of O-sulfate and the presence of N-acetyl or sulfamido groups. Specificity of the heparinase requires the presence of O-sulfate and sulfamido groups while derivatives containing free amino or N-acetyl are not substrates. The heparinase degrades heparitin sulfate to some extent acting apparently on the heparin-like portion.

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Synthesis of heparan sulfate-free chains by human keratinocytes is upregulated during terminal differentiation. The cellular location of this product class and the significance of the differentiation effect are unknown. Differential plasma membrane shearing with cationized colloidal silica was used to evaluate the compartmentalization of the heparan and chondroitin sulfate free chains and their respective proteoglycans in 3T3 fibroblasts and human keratinocytes. The method exploits the topologic segregation of plasma membranes of adherent cells into ventral, dorsal, and intracellular domains and the selective binding of the silica to the dorsal membranes, which by shearing can be separated from ventral membranes adherent to the substratum. Analysis of membrane preparations from sheared cells that had been prelabeled with [35S]-sulfate revealed the proteoglycans to be predominantly ventral, at which location a matrix binding function could be accommodated. Proteoglycans were also recovered from dorsal and intracellular membranes, suggesting active trafficking between intra- and extra-cellular sites. In contrast, the major fraction of heparan and chondroitin sulfate free chains was either cytosolic or associated with intracellular membranes, with the remaining approximately 20% segregated to dorsal and ventral membranes. These results suggest different cellular functions for the proteoglycans and glycosaminoglycan free chains. The partial localization of the free chains to peripheral membranes is compatible with our prior hypothesis that they arise by processing of precursor proteoglycans on cell surfaces. Following this origin, the free glycosaminoglycan polymers could be available to bind ligands such as cytokines prior to transport to intracellular sites of action.

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.

Heparin fractions obtained originally from a variety of biological sources were degraded by Flavobacterial heparinase, and the products obtained were analyzed for structural details. In addition periodate oxidation and nitrous acid degradation were used on the polysaccharides as well as on their enzymatic breakdown products to gain further information about the distribution of uronic acid units and about sulfate substitution. Based on these data an arrangement of lower sulfated regions in an otherwise higher sulfated polymer has been proposed.

Heparitin sulfate has been isolated from several sources, namely: a commercial lung polysaccharide preparation, beef lung, beef aorta, human amyloid liver, human intestine, and urine of a patient with mucopolysaccharidosis. The polysaccharides isolated were extensively purified, fractionated, and characterized. The data obtained show that heparitin sulfate is not a single compound but constitutes a family of related polymers which differ in sulfate content and in the arrangement of charged groups. These compounds are readily distinguished from most other glycosamino-glycuronans (mucopolysaccharides) by composition and optical rotation. They can be differentiated from heparin by their content of sulfate and N-acetyl groups, and D-glucuronic acid residues and by the ratio of the carbazole to orcinol uronic acid values. Due to the variations of charge distribution and molecular size, the heparitin sulfates are found to a considerable extent in admixture with other acidic polysaccharides during isolation and fractionation procedures. The particular type of heparitin sulfate obtained varies considerably with the tissue of origin. The lung-derived material was found to contain the largest range of subfractions. The heparitins isolated from aorta and amyloid liver were fairly homogenous in themselves, but differed from each other.