Wheat germ agglutinin binds to the contact site A glycoprotein of Dictyostelium discoideum and inhibits EDTA-stable cell adhesion

Wheat germ agglutinin (WGA), a lectin that primarily reacts with N-acetylglucosamine residues, specifically inhibits the EDTA-stable type of intercellular adhesion of aggregation competent Dictyostelium discoideum cells. The major WGA-binding protein of these cells is a developmentally-regulated glycolipoprotein of 80 kd apparent mol. wt., designated as contact site A. This glycoprotein is a target site of antibody fragments that block the EDTA-stable cell adhesion, and is characterized by sulfated carbohydrate residues. WGA does not significantly bind to glycoproteins of a mutant, HL220, which produces a 68-kd component in place of the 80-kd glycoprotein. Inhibition of N-glycosylation by tunicamycin causes wild-type cells to produce a WGA-binding but unsulfated 66-kd component and a non-binding 53-kd component. These results indicate that the 80-kd glycoprotein contains two classes of carbohydrate residues, a WGA-binding one that is defective in HL220, and another, sulfated, one that is absent from the 66-kd wild-type product; both are missing in the 53-kd protein. WGA and a monoclonal antibody that is blocked by N-acetylglucosamine were further used to probe for glycoproteins in the multicellular slug stage that share carbohydrate structures - and possibly functions - with the contact site A glycoprotein. Glycoproteins in the 95-kd range have previously been implicated in cell-to-cell adhesion during the slug stage. We distinguished a 95-kd glycoprotein that binds WGA from another one that binds antibody.     

Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies

A series of monoclonal antibodies against a developmentally regulated protein of Dictyostelium discoideum, the contact site A glycoprotein, were used in immunoblots to label proteins of cells harvested at three stages of development: during the growth phase, at the aggregation competent stage, and at the slug stage. The antibodies fell into two groups according to their reactivity with partially or fully deglycosylated forms of the 80 kDa glycoprotein. Group A antibodies reacted not only with a 66 kDa, but also with a 53 kDa product of tunicamycin-treated wild-type cells, and they reacted with a 68 kDa component produced by HL220, a mutant that carries a specific defect in glycosylation. The 68 kDa product of the mutant was not completely unglycosylated. Like the 80 kDa glycoprotein of the wild type, which carried sulfate at carbohydrate residues, the mutant product was sulfated. In the presence of tunicamycin, the mutant produced a 53 kDa component indistinguishable from that of the wild type, which represents, most likely, the non-N-glycosylated protein portion of the contact site A glycoprotein. The group A antibodies showed almost no cross-reactivity with other proteins of the developmental stages tested, in accord with their postulated specificity for the protein moiety of the contact site A molecule. Group B antibodies did not react with the 53 kDa product of tunicamycin-treated cells, nor with the 68 kDa component of mutant HL220. These antibodies were of varying specificity. Some of them were almost as specific as group A antibodies, others cross-reacted with many proteins, particularly of the slug stage. Competition or non-competition between various group B antibodies for binding to the contact site A glycoprotein allowed sub-classification of these antibodies. According to two criteria, group B antibodies were characterized as anti-carbohydrate antibodies: (1) some of these antibodies were blocked by N-acetylglucosamine; (2) none of them reacted with the 68 kDa product or any other protein of mutant HL220. These results indicate that the 80 kDa glycoprotein carries two types of carbohydrate: type 1 carbohydrate that is sulfated and present on the 68 kDa product of mutant HL220, and type 2 carbohydrate that reacts with group B antibodies and is present on the 66 kDa product of tunicamycin-treated wild-type cells. Type 2 carbohydrate moieties are also present on many glycoproteins that are enriched in the prespore area of the slugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.     

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [³⁵S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [³⁵S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [³⁵S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [³⁵S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.     

Intracellular transport and tyrosine sulfation of procollagens V

Several tyrosine residues of the extracellular p-collagens V and collagens V are sulfated [Fessler, L. I., Brosh, S., Chapin, S. and Fessler, J. H. (1986) J. Biol. Chem. 261, 5034-5040]. Here, the sulfation of their intracellular precursors, the procollagens V, was studied. A Golgi-enriched subcellular fraction of chick embryo tendon catalyzed the sulfation of tyrosine residues in both endogenous and added, unsulfated procollagens V with the sulfate donor 3'-phosphoadenosine 5'-[35S]phosphosulfate. Intracellular tyrosine sulfation of procollagen V occurred at a point distal to the cis Golgi compartment as judged by change of the N-linked carbohydrate of procollagen V from being endoglycosidase-H-sensitive to being resistant. The time course of the intracellular modifications of procollagen V was determined by incubating tendons with 3H-labeled amino acids and with [35S]sulfate. The pro alpha(V) chains were synthesised in about 10 min and then assembled into unsulfated procollagen V molecules. Tyrosine sulfation occurred 50 min after completion of polypeptide synthesis and the molecules were successively sulfated in the order in which they had been synthesized. The antimicrotubular drug Nocodazole, which disrupts the spatial organization of the Golgi, decreased the time interval between synthesis of procollagens V and sulfation. The sulfated procollagens V were soon secreted and cut to sulfated p-collagens V. Sulfated pro alpha 1(V) chains were cleaved faster than sulfated pro alpha 1'(V) chains. The relationship of sequential protein modification to spatial cellular organization is discussed.     

Mutants of Dictyostelium discoideum with altered carbohydrate moieties of contact site A.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.     

Murine embryonal carcinoma cell-surface sialyl LeX is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl LeX was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (LeX or X hapten). Sialyl LeX was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl LeX and i antigens but not LeX, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.     

Sulfation of fucoidan in Fucus embryos. I. Possible role in localization

Zygotes of the brown alga Fucus distichus L. Powell divide into two cells which are structurally and biochemically different from each other. Cytochemical staining and autoradiography indicate that a sulfated polysaccharide is localized in only one of the two cells. Up to 10 hr after fertilization, no localization of sulfated polysaccharides is detectable in zygotes, and little ³⁵S (Na₂³⁵SO₄) is incorporated into an acid-soluble carbohydrate fraction. Between 10 and 16 hr, during rhizoid initiation and several hours before the first cell division, there is a large increase in the amount of ³⁵S incorporated into this fraction. The label is found associated with the sulfated fucose polymer fucoidan. Various extraction techniques and labeling experiments demonstrate that fucoidan is unsulfated at fertilization and undergoes little metabolic activity or turnover during the first 24 hr. Thus, the incorporation of sulfate into this carbohydrate fraction appears to involve a sulfation of a preexisting, unsulfated fucan polymer. The degree of sulfation achieved at this time in vivo is sufficient for migration of fucoidan through an electric field in agarose or acrylamide gels. The possible role of sulfation as a mechanism for the localization of fucoidan in the rhizoid cell by means of an intracellular electrical gradient is discussed.     

Identification of carbohydrate moieties involved in EDTA-stable or EDTA-sensitive cell contact of Dictyostelium discoideum

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.     

Chlorate inhibits tyrosine sulfation of human type III procollagen without affecting its secretion or processing

Sodium chlorate, a potent inhibitor of sulfation reactions, completely inhibits the formation of tyrosine-o-sulfate in type III procollagen in human fibroblasts, when used in concentrations that do not affect the incorporation of radioactive amino acids into protein. The unsulfated type III procollagen is secreted into the medium at a rate comparable, to those of sulfated type III procollagen and type I procollagen, which normally does not undergo sulfation. The enzymatic cleavage of the aminoterminal propeptide of type III procollagen is incomplete in fibroblast cultures, irrespective of the sulfation status of the protein.     

Murine embryonal carcinoma cell-surface sialyl Le is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl Le^x was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (Le^x or X hapten). Sialyl Le^x was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl Le^x and i antigens but not Le^x, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.     

Chemoenzymatic synthesis of PSGL-1 glycopeptides: sulfation on tyrosine affects glycosyltransferase-catalyzed synthesis of the O-glycan

PSGL-1 is the primary glycoprotein ligand for P-selectin during the inflammatory response. Interestingly, the N-terminal sequence, containing both a site of tyrosine sulfation and an O-glycan, has been shown to bind to P-selectin with an affinity similar to full-length PSGL-1. To further characterize this system, the synthesis of glycopeptides from PSGL-1 was undertaken. The synthesis involved both solution- and solid-phase synthesis, as well as enzymatic transformations. During the synthesis, notable reactivity differences of the glycosyltransferases toward sulfated and unsulfated versions of the same glycopeptides were observed.     

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.     

Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.     

Cross-reactivity of contact site A to antibody produced against a stage-specific antigen from a phenol/water extract of Dictyostelium discoideum

The stage-specific antigen, gp68 (Hirano, T., Yamada, H., & Miyazaki, T. (1983) Biochim. Biophys. Acta 742, 224-234), was purified from a phenol/water extract of aggregation-competent cells of Dictyostelium discoideum by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Anti-gp68 was produced against purified gp68 which was determined to be homogeneous by silver staining on analysis by SDS-polyacrylamide gel electrophoresis. The cross-reactivity of anti-gp68 against cellular antigens was estimated by immuno-affinity chromatography on anti-gp68 immunoglobulin G (IgG)/Sepharose. When the whole cell lysate was applied to this affinity column, three major proteins, with molecular weights of 80,000, 68,000, and 56,000, were obtained in the absorbed fraction. When the butanol extract, which was enriched in contact site A, an adhesion mediating glycoprotein, was applied to the same column, two major proteins with molecular weights of 80,000 (corresponding to contact site A) and 56,000 were obtained in the absorbed fraction but, however, gp68 was negligible. Reversely, when the phenol/water extract was applied to anti-contact site A-IgG/Sepharose, only gp68 was obtained in the absobed fraction. Moreover, contact site A was seen to compete with [3H]mannose-labeled gp68 in a competition radioimmunoassay using anti-gp68 serum. The effect of Pronase or exo-alpha-mannosidase digestion on the antigenic activity of gp68 was examined by radioimmunoassaying. The results indicated that the alpha-mannosyl residue of the non-reducing terminal in the carbohydrate moiety of gp68 was a major immunodeterminant. However, the polypeptide chain did not participate in the antigenic reactivity against anti-gp68. Both anti-gp68 and anti-contact site A agglutinated heat killed-yeast cells. Also, both anti-sera inhibited EDTA-stable cell adhesion of aggregation-competent cells in the presence of Fab from goat anti-rabbit IgG. These results indicate that gp68 and contact site A have a common antigenic determinant against anti-gp68, and that the target antigen of anti-gp68 was somehow involved in cell adhesion.     

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice

Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides.     

Nonsulfated cholecystokinins in the small intestine of pigs and rats

Cholecystokinin (CCK) is a gut hormone that acts via two receptors. The CCK_A-receptor requires the tyrosyl residue in the C-terminal bioactive site of CCK to be O-sulfated, whereas, the CCK_B-receptor binds irrespective of sulfation. Consequently, unsulfated CCK peptides – if present – may constitute a hormone system that acts only through the CCK_B-receptor. Therefore, we have now examined whether, CCK peptides occur in nonsulfated form in the small intestine of pigs and rats. The concentrations of sulfated and nonsulfated CCK were measured by RIAs, one specific for sulfated CCKs and a new two-step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography.The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat. The porcine duodenal mucosa contained 193 ± 84 pmol/g sulfated CCK and 31 ± 10 pmol/g nonsulfated CCK, and the upper rat intestine 70 ± 19 pmol/g and 8 ± 2 pmol/g, respectively. The degree of sulfation correlated with the endoproteolytic proCCK processing. Thus, 38% of porcine CCK-58 was unsulfated, whereas, only 12% of CCK-8 was unsulfated.The results show that a substantial part of intestinal CCK peptides in rats and pigs are not sulfated, and that the longer peptides (CCK-58 and CCK-33) are less sulfated than the shorter (CCK-22 and CCK-8). Hence, the results demonstrate that proCCK in the gut is processed both to sulfated and unsulfated α-amidated peptides of which the latter are assumed to act via the CCK_B-receptor.     

Papilin: a Drosophila proteoglycan-like sulfated glycoprotein from basement membranes

A sulfated glycoprotein was isolated from the culture media of Drosophila Kc cells and named papilin. Affinity purified antibodies against this protein localized it primarily to the basement membranes of embryos. The antibodies cross-reacted with another material which was not sulfated and appeared to be the core protein of papilin, which is proteoglycan-like. After reduction, papilin electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band of about 900,000 apparent molecular weight and the core protein as a narrow band of approximately 400,000. The core protein was formed by some cell lines and by other cells on incubation with 1 mM 4-methylumbelliferyl xyloside, which inhibited formation of the proteoglycan-like form. The buoyant density of papilin in CsCl/4 M guanidine hydrochloride is 1.4 g/ml, that of the core protein is much less. Papilin forms oligomers linked by disulfide bridges, as shown by sodium dodecyl sulfate-agarose gel electrophoresis and electron microscopy. The protomer is a 225 +/- 15-nm thread which is disulfide-linked into a loop with fine, protruding thread ends. Oligomers form clover-leaf-like structures. The protein contains 22% combined serine and threonine residues and 25% combined aspartic and glutamic residues. 10 g of polypeptide has attached 6.4 g of glucosamine, 3.1 g of galactosamine, 6.1 g of uronic acid, and 2.7 g of neutral sugars. There are about 80 O-linked carbohydrate chains/core protein molecule. Sulfate is attached to these chains. The O-linkage is through an unidentified neutral sugar. Papilin is largely resistant to common glycosidases and several proteases. The degree of sulfation varies with the sulfate concentration of the incubation medium. This proteoglycan-like glycoprotein differs substantially from corresponding proteoglycans found in vertebrate basement membranes, in contrast to Drosophila basement membrane laminin and collagen IV which have been conserved evolutionarily.     

In vivo and in vitro tyrosine sulfation of a membrane glycoprotein

A431 cells incorporate 35SO4 into a protein of Mr 61,000 (P61). We examined sulfation of P61 by cells (in vivo) and by a cell-free system (in vitro) which requires only addition of A431 cell membranes and a 3'-phosphoadenosine 5'-phospho[35S]sulfate-generating system prepared from Krebs ascites cells. Sulfate is found exclusively in the form of tyrosine SO4 by two-dimensional high voltage electrophoresis following Pronase digestion. Endoglycosidase F digestion reduces the Mr by 2,000 but does not release the sulfate, indicating that P61 is a glycoprotein but that sulfate is not incorporated into the carbohydrate. Sulfated P61 is not found in the medium from cultured cells and remains associated with the membrane fraction following cell lysis. Treatment of membranes with 0.4 M NaCl, 0.3 M KCl, 15 mM EDTA, or pH 11.0 does not release sulfated P61. P61 is solubilized by Triton X-114 treatment of membranes and partitions into the detergent phase upon warming. Based on these characteristics, we conclude that P61 is an integral membrane protein. Trypsin digestion experiments with intact cells suggest that sulfated P61 is predominantly located in the plasma membrane. This is the first example of an integral membrane protein which is sulfated on tyrosine. The properties of the sulfation reaction are distinct from those reported for secreted proteins and are consistent with the possibility that this modification occurs at the plasma membrane rather than in the Golgi.