Differential Regulation of Kidney and Spleen Cytokine Responses in Mice Challenged with Pathology-Standardized Doses ofCandida albicansMannosylation Mutants

ABSTRACTCell surface polysaccharides are key determinants of host responses to fungal infection. We determined the effects of alterations inCandida albicanscell surface polysaccharide composition and gross changes in the host immune response in groups of mice challenged intravenously with fiveC. albicansstrains at doses adjusted to give equal disease progression 3 days later. The five strains used were the parental strain NGY152, two mutants with defective cell wall mannosylation,pmr1Δ mutant andmnt1/2Δ mutant, and the same two strains with a copy ofPMR1andMNT1reintegrated, respectively. Renal and spleen levels of chemokines and cytokines previously shown to be key components of early host response toC. albicanswere determined at intervals up to 3 days after challenge. By 12 h afterC. albicanschallenge, the levels of granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), interleukin 6 (IL-6), monocyte chemotactic peptide 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and MIP-2 were higher in the kidneys of mice challenged with thepmr1Δ mutant than in animals challenged with the other strains and were lower by day 3, suggesting an earlier host response to thepmr1Δ mutant. The production of these chemokines also diminished earlier than controls in mice infected with themnt1/2Δ strain. Although these differences were statistically significant, their magnitude was seldom great, and no unambiguous evidence was obtained for individual responses specific to any cell surface glycosylation change. We conclude that complex, multifactorial local responses offset and obscure any differences resulting from differences in surface mannosylation ofC. albicansstrains when infection results from pathology-standardized challenges.

Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (Kd = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.

A tumor-associated glycosylation change in the glucose transporter GLUT1 controlled by tumor suppressor function in human cell hybrids

Studies of human cell hybrids have provided evidence that the tumorigenicity of a cervical carcinoma (HeLa) is under the control of a putative tumor suppressor on chromosome 11. Using these human cell hybrids, we found a tumor-associated glycosylation change in the glucose transporter GLUT1, which is an N-linked glycoprotein at the plasma membrane. The non-tumorigenic HeLa × fibroblast cell hybrid CGL1 and the normal diploid fibroblast WI38 expressed the 50–55 kDa GLUT1, whereas in a tumorigenic segregant hybrid, CGL4, as well as in parental HeLa cells, GLUT1 glycosylation was altered and its molecular mass was about 70 kDa. However, the altered GLUT1 glycosylation was not observed in SV40-transformed WI38 cells, suggesting a correlation between this glycosylation change and a putative tumor suppressor function. Further investigations using glycosidases, glycosylation inhibitors and lectin-affinity chromatography demonstrated that the tumor-associated glycosylation change in GLUT1 was mainly due to the increase in N-acetyl-lactosamine repeats in the N-linked oligosaccharides. In accordance with the altered glycosylation, affinity for 2-deoxyglucose in the tumorigenic CGL4 cells increased 2-fold, but there was little change in the Vmax. These results suggest there may be a functional role for the modulation by glycosylation of GLUT1 in the tumorigenic behavior of CGL4 and HeLa cells.