Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.     

Decreased ganglioside neuraminidase activity in fibroblasts from mucopolysaccharidosis patients. Inhibition of the activity in vitro by sulfated glycosaminoglycans and other compounds

The neuraminidase activities towards the ganglioside substrates GD1a, GD3 and GM3 were found to be markedly diminished in homogenates of fibroblasts cultured from patients with various genetic mucopolysaccharidoses. Mixing normal and patients' fibroblast homogenates revealed this effect to be due to the presence of diffusible inhibitors. The neuraminidase acting on the trisaccharide sialyllactose, on the other hand, showed normal activity in all the cell lines tested. Experiments in vitro revealed the sulfated glycosaminoglycans chondroitin 4-sulfate and heparin, the polysaccharide dextran sulfate, and the trypanocidal drug suramin to be strongly inhibitory on the ganglioside GD1a neuraminidase activity of normal fibroblast homogenates. Regarding chondroitin 4-sulfate, this inhibition was of the non-competitive type. A disulfated tetrasaccharide prepared from chondroitin 4-sulfate, on the other hand, was not at all inhibitory. These and additional findings led us to propose a model for the interaction between enzyme and inhibitor, involving a 'clamping' mechanism by the polysulfated compounds. We conclude that the decreased ganglioside neuraminidase activities of mucopolysaccharidosis fibroblasts are due to an inhibition by the accumulated sulfated glycosaminoglycans and that such inhibition is responsible for the storage of certain gangliosides in the tissues of the patients.     

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.     

Characterization of glycosaminoglycans stored in mucopolysaccharidosis III A: evidence for a generally occuring degradation of heparan sulfate by endoglycosidases.

The characterization of intracellularly stored glycosaminoglycans from organs of a patient suffering from mucopolysaccharidosis III A (Sanfilippo A disease) is described. Both heparan sulfate and galactosamine-containing glycosaminoglycans (chondroitin sulfate, dermatan sulfate) are accumulated in the liver, whereas in the other organs (spleen, kidney, heart, cerebrum, cerebellum) heparan sulfate is almost the only glycosaminoglycan stored. It is shown by [3H]NaBH4 reduction and subsequent identification of the 3H-labelled sugar alcohols that heparan sulfate is degraded in all organs by at least two endoglycosidases, an endoglucuronidase and an endoglucosaminidase, to fragments of low molecular weight (Mr approximately 2 000-6 600).     

Mucopolysaccharidosis Types IH, IS, II and IIIA: Glycosaminoglycans and Lipids of Isolated Brain Cells and Other Fractions from Autopsied Tissues

Abstract: Brain cellular fractions were prepared in bulk from four non-neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8-month-old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one-half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four- to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides G_{m 2}, G_{m 3}, G_{d 3} and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of G_{m 3}, G_{m 2} and G_{d 3} together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.     

Urinary excretion of glycosaminoglycans and albumin in experimental diabetes mellitus

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.     

Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4

Platelet factor 4 (PF-4) is a platelet-derived alpha-chemokine that binds to and activates human neutrophils to undergo specific functions like exocytosis or adhesion. PF-4 binding has been shown to be independent of interleukin-8 receptors and could be inhibited by soluble chondroitin sulfate type glycosaminoglycans or by pretreatment of cells with chondroitinase ABC. Here we present evidence that surface-expressed neutrophil glycosaminoglycans are of chondroitin sulfate type and that this species binds to the tetrameric form of PF-4. The glycosaminoglycans consist of a single type of chain with an average molecular mass of approximately 23 kDa and are composed of approximately 85-90% chondroitin 4-sulfate disaccharide units type CSA (-->4GlcAbeta1-->3GalNAc(4-O-sulfate)beta1-->) and of approximately 10-15% di-O-sulfated disaccharide units. A major part of these di-O-sulfated disaccharide units are CSE units (-->4GlcAbeta1-->3GalNAc(4,6-O-sulfate)beta1-->). Binding studies revealed that the interaction of chondroitin sulfate with PF-4 required at least 20 monosaccharide units for significant binding. The di-O-sulfated disaccharide units in neutrophil glycosaminoglycans clearly promoted the affinity to PF-4, which showed a Kd approximately 0.8 microM, as the affinities of bovine cartilage chondroitin sulfate A, porcine skin dermatan sulfate, or bovine cartilage chondroitin sulfate C, all consisting exclusively of monosulfated disaccharide units, were found to be 3-5-fold lower. Taken together, our data indicate that chondroitin sulfate chains function as physiologically relevant binding sites for PF-4 on neutrophils and that the affinity of these chains for PF-4 is controlled by their degree of sulfation.     

The Circulating Glycosaminoglycan Signature of Respiratory Failure in Critically Ill Adults

Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.     

Effect of DEAE-dextran and dextran sulfate on [35S]sulfate incorporation into glycosaminoglycans of normal and SV40 virus-transformed 3T3 cells.

Short (10 min) and long (24 hr) treatments with 10 μg/ml dextran sulfate produced a marked increase in the incorporation of [³⁵S] sulfate into cell-associated and extracellular glycosaminoglycans of SV40-transformed 3T3 cells. Similar treatment of 3T3 cells did not affect glycosaminoglycan synthesis. DEAE-dextran treatment for 24 hr produced a large increase in the cell-associated glycosaminoglycans and a proportional decrease in the extracellular sulfated glycosaminoglycans. The types of glycosaminoglycans synthesized were not affected significantly by the treatments.     

Age-related changes in the content and composition of glycosaminoglycans isolated from the mouse skeletal muscle: normal and dystrophic conditions

Glycosaminoglycans were isolated from the skeletal muscle of either normal or dystrophic mice aged from 3 to 18 weeks. The glycosaminoglycan content of the normal muscle, based on the tissue weight, decreased slightly during the period from 3 to 10 weeks, and remained almost unchanged after 10 weeks. The major glycosaminoglycan in normal muscle was hyaluronate, the relative amount of which increased slightly (from 70% to 80%) with age. Both dermatan sulfate and heparan sulfate were also obtained. The relative amounts of these sulfated glycosaminoglycans tended to decrease with age. On the other hand, the glycosaminoglycan content of the dystrophic muscle was higher than that of normal muscle even at 3 weeks. The proportion of hyaluronate was almost constant (about 65%) throughout the age range examined. The relative amount of dermatan sulfate increased from 20% to 30% with a compensatory decrease in the amount of heparan sulfate. Further, the incorporation of [35S]sulfate into glycosaminoglycans by the dystrophic muscle was reduced to about 60% of the normal. These differences in glycosaminoglycan composition and [35S]sulfate incorporation between the normal and the dystrophic muscles may be related to the progressive muscular dysfunction seen in this disease.     

Stimulation of proteoglycan synthesis in chick embryo sternum by serum and L-3,5,3'-triiodothyronine.

Incorporation of sulfate into alcian blue-precipitable glycosaminoglycan of 12-day-old chick embryo sterna is stimulated by addition, separately or together, of normal human serum and physiological concentrations of thyroid hormones (Audhya, T.K., and Gibson, K.D. (1975) Proc. Natl. Acad, Sci. U. S. A. 72, 604--608). We present evidence that this stimulation is due to increased synthesis of at least one proteoglycan, with minor alterations in the size and chemical composition of the glycosaminoglycans. Pulse-chase experiments showed no detectable loss of label during the chase, in control sterna or sterna incubated with serum and L-3,5,3'-triiodothyronine; thus, all incorporation was the result of synthesis of glycosaminoglycans. In double-label experiments, with 35SO4(2-) and [3H]acetate, the molar ratio of 3H and 35S incorporated into glycosaminoglycans was changed little, if at all, by addition of serum or triiodothyronine or both, at concentrations which increased incorporation up to 2-fold. Glycosaminoglycans isolated from these and other incubations gave similar elution patterns from agarose columns, and identical electrophoretic patterns on cellulose acetate. Digestion with chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4.) showed that incorporation was into chondroitin sulfate and possibly hyaluronic acid, and that the proportions of non-sulfated, 4-sulfated, and 6-sulfated disaccharide units differed little between stimulated and unstimulated sterna. Incorporation of [3H]serine into glycosaminoglycans from papain digest of sterna paralleled incorporation of 35SO4(2-), and indicated a number average molecular weight between 21,000 and 25,000 for the newly synthesized chondroitin sulfate. This value was confirmed by gel filtration chromatography, which also showed that the average molecular weight of the newly synthesized chondroitin sulfate decreased up to 15% under conditions of 2-fold stimulation. Proteoglycans were extracted from sterna incubated with [3H]serine and 35SO4(2-) and analyzed by isopycinic centrifugation in CsCl and by zone sedimentation in a sucrose gradient. A major proteoglycan fraction could be separated by either method. Incorporation of both isotopes into this proteoglycan fraction, and into glycosaminoglycans isolated after papain digestion, was stimulated in a coordinate manner. Almost identical results were obtained with both separation techniques. The results indicate that the synthesis of the major proteoglycan, and probably also of a minor one, is stimulated by serum and triiodothyronine.     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

The effect of sexual hormones on the sulfated glycosaminoglycan pattern of male genital accessory organs

The absolute and relative amounts of glycosaminoglycans and [35S]sulfate uptake were investigated in several tissues of male guinea pigs and rats under different sexual hormonal conditions (castration, estrogen treatment, or both). The hormonal effects, regarding the pattern of sulfated glycosaminoglycans, were specifically observed in the target organs (vas deferens and seminal vesicles) of both animals. Castration, in both species, decreases the amount of heparan sulfate and chondroitin sulfate, while diethylstilbestrol (DES) treatment causes different effects on rat and guinea pig target organs. In rats the effect of estrogen administration and surgical castration was essentially the same, and in guinea pigs DES increased the content of dermatan sulfate and chondroitin sulfate. The modifications in the specific patterns of the sulfated glycosaminoglycans suggest that these compounds are under sexual hormonal control only in the target organs, and show a specific pattern of distribution according to the tissue layer.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteoglycan synthesis in normal and Lowe syndrome fibroblasts

Lowe (oculocerebrorenal) syndrome (LS) is an X-linked disorder characterized by congenital cataracts, generalized hypotonia, mental retardation, and renal Fanconi syndrome. The basic defect remains unknown, but the possibility that fibroblasts express reduced sulfation of glycosaminoglycans has been studied in several laboratories. A mechanism involving overproduction of an enzyme (nucleotide pyrophosphatase) active against adenosine 3'-phosphate, 5'-phosphosulfate (PAPS) has been postulated. Decreased synthesis of normally sulfated glycosaminoglycans was also reported. We measured the synthesis of proteoglycans and glycosaminoglycans by incorporation of [3H]glucosamine and Na2(35)SO4 into cultured fibroblasts from four LS patients and related it directly to the synthesis in six normal fibroblast cultures. We found that the rate of synthesis varied greatly among the normal cultures (cv, 30%), but not significantly between LS and the normal. The LS fibroblasts' ability to sulfate glycosaminoglycans was assayed as the amount of 3H-glycosaminoglycan eluting at low ionic strength on anion exchange chromatography, the amount of non-sulfated disaccharide present in chondroitinase digests of labeled proteoglycans, and the ratio of 35S to 3H incorporation into proteoglycans. Each parameter suggested that the LS cells were synthesizing normally sulfated glycosaminoglycans (e.g. % delta Di-0S, 21 +/- 6 in normal; 27 +/- 6 in LS). The cells' ability to sulfate glycosaminoglycans was tested under conditions of markedly stimulated glycosaminoglycan synthesis, by treating the cultures with a beta-D-xyloside. LS and normal cells responded to the treatment by elevating the rate of synthesis of normally sulfated glycosaminoglycans (3.5-6-fold in normal, 3-7-fold in LS). Nucleotide pyrophosphatase activities were found to be elevated in each of our four LS cell strains as in the previous studies, excluding genetic heterogeneity as an explanation for our findings. We conclude that LS fibroblasts do not express defects in sulfation of glycosaminoglycans or in synthesis of proteoglycans.     

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig

The majority of glycosaminoglycans synthezied in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment, indicating that the glycosaminoglycanas existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine, Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicate that ΔDi-diS_E derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [³⁵S]sulfate and [³H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the relese of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.