Oligomannosides initiate cell spreading of laminin-adherent murine melanoma cells.

Murine melanoma cells readily bind and spread on murine laminin. Uncoupling of spreading from adhesion occurs when unglycosylated laminin is used as the cellular substratum; spreading is restored by soluble glycosylated laminin or soluble glycopeptides of laminin. In this study, using kifunensine, we produced and characterized an oligomannoside-rich glycoform of laminin. When used as a substratum for cell attachment and spreading, this laminin was as effective as mature glycosylated laminin. When added in solution to unglycosylated laminin-adherent cells, kifunensine-laminin was more effective in promoting cell spreading than mature glycosylated laminin. Reconstitution with soluble polysaccharides showed that cell spreading was initiated rapidly by microgram amounts of mannan but not other polysaccharides and approached a maximum within 1 h; titration with mannan yielded an adsorption isotherm profile. Mannose was an antagonist, preventing mannan from restoring cell spreading, but it was not an agonist. A Pronase digest of mature glycosylated laminin, depleted of its oligomannoside-peptides, was unable to restore cell spreading, whereas a control digest was fully active. Melanoma cells were unable to bind to three different neoglycoprotein surfaces, but when soluble unglycosylated laminin was present in the medium the cells adhered to and spread only upon mannosylated bovine serum albumin. Of the various cell lines known to interact with glycosylated laminin only murine melanoma cells showed oligomannoside-dependent spreading on an unglycosylated laminin substratum. The composite results indicate that oligomannosides are necessary to initiate murine melanoma cell spreading on laminin but are not sufficient for cell adhesion.

Characterization of oligomannoside binding to the surface of murine melanoma cells. Potential relationship to oligomannoside-initiated cell spreading.

The spreading of murine melanoma cells on unglycosylated laminin is initiated by soluble glycosylated laminins containing oligomannosides, or by mannan, and is inhibited by mannose (Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3356-3366). In the present study, melanoma cell recognition of oligomannosides was explored by several different methods. Comparison of cell spreading, initiated by related branched oligomannosides, showed that micromolar concentrations of Man6 and Man9 were able to restore cell spreading maximally, whereas Man3 was ineffective. Man9 bound to the melanoma cells in a bimodal manner at micromolar concentrations, reaching saturation, whereas Man3 showed linear binding in the same and higher ranges. Comparison of Man9 binding with Man9-initiated cell spreading demonstrated that maximal spreading occurred at approximately half-saturation. Competition experiments showed that mannose or mannan effectively impaired Man9 binding to the cells, whereas galactose or fucose was ineffective. Mannan-conjugated fluorescent beads bound in a diffuse pattern to the surface of melanoma cells, whereas underivatized beads did not bind. The distribution of laminin-binding beta 1 integrin on the cell surface was compared with surface mannan binding. The beta 1 integrin staining pattern did not match the mannan staining pattern either in attached, nonspread cells or in attached, spread cells. The composite results support a model in which occupancy of both an integrin and a cell surface lectin is required for murine melanoma cells to spread on glycosylated laminin.

Postextraction osteomyelitis in a bone marrow transplant recipient.

This report describes a case of mandibular osteomyelitis after a dental extraction in a patient who subsequently underwent bone marrow transplantation (BMT) for lymphoblastic lymphoma. Surgical guidelines consistent with National Cancer Institute recommendations were followed for the extraction, which was performed before initiation of the myelosuppressive conditioning regimen. However, moderate tenderness developed at the extraction site beginning 10 days after marrow infusion. On day 26 the patient became febrile and blood culture-positive for Staphylococcus epidermidis. Radiographs exposed on day 28 demonstrated changes consistent with low-grade osteomyelitis, including diffuse loss of lamina dura and an irregular osseous rarefaction extending 1 cm posterior to the extraction site. Although the indwelling Hickman catheter was the presumed source for bacteremia, clinical and radiographic data led to consideration of mandibular osteomyelitis as an alternative cause. Characteristics of this infection in BMT recipients are reviewed. Recommendations for dental extractions and prophylactic antibiotic regimens for catheterized BMT recipients are also discussed. Although mandibular osteomyelitic lesions are not common in profoundly immunosuppressed BMT recipients, prompt recognition and treatment are essential when the disease occurs.

Laminin oligosaccharides play a pivotal role in cell spreading.

The basement membrane glycoprotein laminin promotes cell adhesion, spreading and neurite outgrowth. We can uncouple cell adhesion and spreading (or neurite outgrowth) when unglycosylated laminin is used as a substratum. Mouse melanoma cells, B16F1 line, readily attach to unglycosylated laminin but fail to spread once adherent. Spreading can be restored by titration with glycosylated laminin or with laminin glycopeptides. When the laminin substratum is absent in the test chambers, the cells do not adhere when either intact laminin or its glycopeptides are then added. Analyses show that these added substances are recoverable from the culture medium and do not bind to the chamber surfaces. Use of selective inhibitors which interfere with carbohydrate processing yields several glycoforms of laminin which we have isolated and examined for their ability to support cell adhesion and spreading. Laminin which is enriched in high mannose oligosaccharides is much more effective in promoting cell spreading than laminin which is enriched in hybrid oligosaccharides. These results are consistent with earlier studies which showed that ConA, which primarily recognizes mannose residues, could also uncouple cell adhesion and spreading. Although mono- and disaccharides failed to restore cell spreading, we have found that addition of various mannose oligosaccharides to adherent cells effectively reestablishes their spreading behavior. The extent of cell spreading which is achieved by the added saccharides is related to their amount, their duration of addition, and their molecular structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of laminin carbohydrates on cellular interactions.

Laminins, a family of large multidomain glycoproteins of the basal lamina, have been implicated in the development and maintenance of cellular and tissue organization. Considerable interest has arisen concerning the ways in which laminin carries out its biological functions. Previously these biologic responses have been primarily attributed to the peptide sequences of laminin, however, newer studies suggest that laminin carbohydrates may also participate in such cellular activities. Recently, a subpopulation of laminin molecules purified from EHS sarcoma by lectin affinity chromatography has been shown to contain about 25 to 30% carbohydrate. Most of the carbohydrates present are complex-type asparagine-linked oligosaccharides encompassing many different structures, some of which are unique to laminin. To date, the biological function of the carbohydrates of laminin remains somewhat unclear. They do not appear to be needed for heparin binding or to enhance proteinase stability, however, current evidence suggests they are important in cellular spreading and neurite outgrowth. It is our hypothesis that in the covalently-linked carbohydrate moieties of laminin will ultimately prove to be involved in information transfer to responsive cells. It is the purpose of this review to delineate current concepts of the structure and function of this unique glycoprotein's sugar chains.

Laminin carbohydrates are implicated in cell signaling.

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553-12562). In the present experiments, we determined whether those cellular responses could be restored to adherent cells. When a mixture of unglycosylated and glycosylated laminins was used as a substratum for mouse melanoma cells, some cells began to spread when 30% glycosylated laminin was present. At least 65% glycosylated laminin was required to elicit a maximal spreading response by the majority of the cells. In separate experiments, we found that cell spreading was fully restored by a pronase digest of glycosylated laminin; a similar digest of unglycosylated laminin had no effect. These results indicate that laminin carbohydrates, rather than polypeptide sequences, were responsible for cell spreading. We also conclude that substrate attachment of the carbohydrate moieties was not essential. In other experiments, laminins containing immature oligosaccharides were produced using two glycosylation pathway inhibitors, swainsonine or castanospermine. When such laminins were used to study cell spreading or neurite outgrowth, laminin containing immature oligosaccharides was as effective as laminin which contains fully processed oligosaccharides. In contrast, laminin with partially processed oligosaccharides had incomplete activity. These composite reconstitution experiments show that laminin carbohydrates provide essential information to responsive cells, enabling them to progress from an adherent state to a spread form or to extend neurite processes.

A human periodontal ligament fibroblast clone releases a bone resorption inhibition factor in vitro.

The conditioned media (CM) obtained from three lines of cloned human periodontal ligament (PDL) cells were analyzed to determine whether they altered the parathyroid hormone (PTH)-stimulated resorption rates (45Ca release) in 48-hour cultures of 45Ca-labeled rat long bones. One PDL cell line, PDL-5, produced a heat-resistant factor in its CM that inhibited the PTH-stimulated resorption by 43.8 +/- 9.7 (SE) percent (p less than or equal to 0.02), whereas the CM from the other cell lines were without statistically significant effect. The CM from the PDL-5 line did not diminish organ culture viability, as determined by 3H-thymidine incorporation, and did not enhance or diminish the resorption-inhibiting activity of calcitonin added to the PTH-stimulated cultures. The addition of CM from PDL-5 did not alter the bone-resorbing effect of interleukin-1 (IL-1). These results indicate that CM from PDL-5 inhibits only the PTH-induced and not the IL-1-mediated resorption processes, whose mechanisms are therefore likely to differ.