Fungal Mannoproteins: the Sweet Path to Immunodominance Mannose residues on glycoproteins trigger key host immune responses that, if better harnessed, could protect against damage and disease
Michael K. Mansour and Stuart M. Levitz
Stuart M. Levitz is a Professor of Medicine and Microbiology at Boston University School of Medicine and Michael K. Mansour is an M.D.-Ph.D. student at Boston University School of Medicine, Boston, Mass.
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Protein tertiary and quaternary structures often depend on sugar residues that are added to newly translated polypeptide chains while they reside in the endoplasmic reticulum (ER) and Golgi apparatus of eukaryotic cells. The ability to decorate proteins with carbohydrates is a function found in more highly evolved eukaryotic organisms such as mammals, fungi, and parasites. In contrast to eukaryotic cells, bacteria, with few exceptions, do not produce complex carbohydrate-modified proteins.
Various eukaryotic species differ in terms of how they use carbohydrates to modify cellular proteins. For example, mammalian immune systems recognize fungal glycoproteins as foreign, primarily based on exposed mannose residues within those proteins. Because this response might provide a means to protect against harmful infections, we are studying mannosylated glycoproteins or mannoproteins (MP) from the pathogenic yeast Cryptococcus neoformans and analyzing how the immune system recognizes and responds to them.
Those mannose residues on MPs serve as ligands for powerful endocytic lectin receptors on antigen-presenting cells (APCs). This foreign patterning of carbohydrate residues is recognized, enabling rapid entry of MP into APCs, in which this glycoprotein is directed to compartments specialized in degrading and processing engulfed antigens. Subsequently, peptide fragments of MP are presented on major histocompatibility complex (MHC) molecules to stimulate T cells. This process very much depends on the initial interaction of MP with those lectin receptors and can be blocked if MP is stripped of its mannose sugars. Such findings suggest that this is the major route by which MPs enter APCs and that this lectin-carbohydrate recognition system contributes significantly to host immune responses.
Fungi and Mammalian Cells Differ in How They Decorate Proteins With Sugars
Fungi provide a relatively simple means for analyzing posttranslational protein glycosylation. Eukaryotic cells have at least three mechanisms by which they attach carbohydrates to nascent proteins: (i) adding glycosylphosphatidylinositol (GPI) anchors, (ii) glycosylating through amino acids containing an amino side chain (N-linked glycosylation), and (iii) glycosylating through a hydroxyl-containing amino acid of the protein (O-linked glycosylation). Fungi and mammalian cells contain all three of these types of glycosylated proteins. In mammalian cells, however, GPI anchors tether proteins to cell membranes, whereas in fungal cells GPI anchors are also used to covalently link proteins to cell wall glucans.
The lipid carrier dolichol is involved in forming N-linked carbohydrate modifications within the ER of eukaryotic cells. When nascent proteins are translated into the ER, a universal carbohydrate core is transferred from dolichol to the protein via an oligosaccharyltransferase complex. In the case of N-linked modifications, the carbohydrate block is accepted by specific asparagines in the recipient protein. These modifications usually occur within the consensus sequence Asn-X-Ser/Thr, where X is any amino acid except proline.
This transfer appears to occur identically in fungal and mammalian cells. Characteristic differences between fungal and mammalian glycoproteins arise because of trimming and regrowth of carbohydrate branches following transfer of nascent glycoproteins to the Golgi body in the respective cell types. Thus, fungi tend to lightly trim core carbohydrate blocks within glycoproteins, leaving a high-mannose core that consists of at least eight or nine mannose residues. Branches from this core can then extend to reach more than 200 monosaccharides in length. Such elongated forms, which are often referred to as mannan, phosphomannan, or galactomannan, differ among fungal species. Meanwhile, within the Golgi body of mammalian cells, most mannose residues are removed from glycoproteins.
N-linked saccharides may also be modified to contain net charges. For example, some N-linked glycoproteins of yeast carry negative charges when they acquire phosphate groups, whereas negatively charged, N-linked mammalian glycoproteins typically contain sialic acids. The lengths of the branching saccharides vary in terms of the type of linkage, length, and selection of monosaccharide. Although rare sugars such as xylose can be found, the most common monosaccharide used by fungi is mannose by itself or in combination with galactose. In contrast, mammalian cells rarely contain mannose as an N-linked extension.
Both mammalian and fungal glycoproteins also may contain O-linked sugars that are attached through either serine or threonine residues and typically contain only two or three saccharide units. The chief difference between mammalian and fungal O-linked glycoproteins depends on the type of monosaccharide that each contains. Thus, mammalian glycoproteins typically include an initial unit of N-acetylgalactosamine (GalNAc) along with combinations of galactose, fucose, GlcNAc, or the more complex sialic acid N-acetyl neuraminic acid (Neu5Ac, also abbreviated as NeuNAc or NANA). Meanwhile, fungi O-linked glycoproteins contain an initial unit of mannose along with additional units of mannose by itself or in combination with galactose.
Although O-linked saccharides on fungal glycoproteins tend to be considerably shorter than those added as N-linked modifications, the former class of glycoproteins appears to be considerably more abundant and therefore may account for the majority of sugars. The O-linked mannoses connect via alpha (1,2)- or alpha (1,3)-linkages to the protein, and typically are extended into linear chains containing two or three saccharide residues, but may contain as many as five such residues in Saccharomyces cerevisiae and as many as seven in Candida albicans. Moreover, both branching and phosphorylation of O-linked glycans have been described.
O-linked and N-linked modifications of fungal proteins appear to be essential for proper folding and intracellular trafficking of the glycoproteins. In addition, the glycan components are necessary for cell wall stability, morphogenesis, resistance to antifungal compounds, and perhaps also virulence. Because the glycoproteins and the biosynthetic pathways for glycans in fungal and mammalian cells differ markedly from one another, the fungal glycoproteins may prove to be valid targets for immunological interventions, while the biosynthetic pathway may prove to be a useful target for the development of antifungal products. With regard to the latter, the pradimicins are a class of antifungal compounds that target fungal mannoproteins and are undergoing preclinical testing.
Cryptococcal Mannoproteins as Potential Protective Antigens
C. neoformans is a yeast pathogen with worldwide prevalence. Its most notable feature is a large polysaccharide capsule with powerful immunoinhibitory properties. Although many individuals may be exposed to C. neoformans, those who are suffering from defects in CD4+ T cells are at a far higher risk for becoming infected. The prevalence of cryptococcosis is particularly high in individuals receiving immunosuppressive agents, such as transplant recipients, and in those infected with HIV. Because T-cell immune responses are critical for clearing C. neoformans infections, individuals with T-cell deficiencies may develop life-threatening complications, including meningoencephalitis.
A group of cryptococcal MP elicits potent T-cell responses in mice that are infected with C. neoformans. Such animals develop delayed-type hypersensitivity reactions, and their T cells proliferate and become activated in vitro, producing interleukin-2 (IL-2). We find that vaccinating mice with MP partially protects them against challenges with C. neoformans. Similarly, T cells obtained from patients who have recovered from cryptococcal infections proliferate when stimulated with MP. MP thus can stimulate human monocytes as well as murine macrophages to induce IL-12 and tumor necrosis factor-alpha (TNF-alpha), two cytokines that play essential roles in mice responding to cryptococcal infections. Cryptococcal MP make up a group of proteins whose molecular sizes range from 20 to more than 100 kDa. While most immunological responses to MP are proinflammatory, a partially purified MP fraction from C. neoformans, designated MP-4, desensitizes neutrophils to this pathogen. Nevertheless, other components from this heterogeneous group make attractive vaccine candidates.
We recently cloned and sequenced the genes encoding two such MP, designated MP98 and MP88, both of which stimulate T-cell responses in immunized mice. The MP N-terminus DNA sequence encodes a secretion signal peptide that presumably enables MP to be transported outside the cell membrane. Consistent with this possibility, MP constitute approximately half the measurable proteins in the supernatant of growing cryptococcal cultures.
The C-termini of these two MP genes encode similar domains containing many serine and threonine residues, with each followed by a potential GPI-anchor motif. For MP98, the serine/threonine rich region stretches 53 amino acids and contains 34 (64%) serine and threonine residues. By genomic analysis, more than 10 other putative cryptococcal MP were identified that are also likely to contain N-terminal signal sequences and C-terminal regions that are rich in serine and threonine, each followed by GPI anchors (Fig. 1).
Apart from the N- and C- termini, the cryptococcal mannose proteins contain very different amino acid sequences. For example, MP98 contains an enzymatic motif that resembles those found in chitin deacetylases. The MP88 amino acid sequence is similar to one found in an allergen from the skin fungus Malassezia furfur. However, it contains no domains of recognizable function.
The cryptococcal MP appear to be heavily glycosylated, with long stretches rich in serine and threonine residues to serve as sites for O-linked saccharides. For instance, when we analyzed cryptococcal MP, they contain predominantly carbohydrate rather than protein. Moreover, after SDS-polyacrylamide gel electrophoresis, MP bands react strongly with carbohydrate-specific stains. When MP are chemically treated to remove O-linked saccharides, their apparent molecular sizes shift, and they no longer stain positive for carbohydrates. Moreover, the MP appear to contain mannose as terminal sugars, as determined by staining with the plant lectin concanavalin A.
Other Mannoproteins Elicit Immune Responses
Host Receptors Can Recognize Foreign Carbohydrates
Having made inroads to determining the molecular structure of cryptococcal MP, we sought to determine how they stimulate T-cell responses. As a general rule, T cells cannot be stimulated without the assistance of antigen presentation, a function reserved for APCs. This process involves capturing foreign material such as whole microorganisms or particular molecules, degrading them within lysosomal vacuoles, and eventually loading them onto a variety of surface-bound presenting molecules, including MHC class II.
These antigen-presenting molecules, in turn, maintain a stable platform on which a T cell, through its specialized T cell receptor (TCR), binds and, in effect, examines the loaded fragment from a foreign protein or cell structure. If a T cell recognizes such an antigen, the cell can become activated. Among three types of distinct APCs--namely, macrophages, B cells, and dendritic cells (DC)--only the latter can activate a naive T cell. The other APCs, especially macrophages, assume mainly maintenance and supportive roles as part of cell-mediated immunity. Therefore, identifying immunodominant molecules that target APCs is a priority because they play such a central role in stimulating T cells that protect the host.
We hypothesized that, because it is extensively decorated with mannose residues, MP could be critical for eliciting vigorous T-cell responses. We know that those sugars promote the efficient uptake, processing, and antigen presentation of MP by APCs. In particular, exposed terminal mannoses on the glycyl sidechains of MP stimulate a strong immune response.
On a molecular level, pattern recognition receptors (PRR) on the surfaces of APCs recognize molecules from pathogens. In this case, lectin receptors form a subset of PRRs that bind specific configurations of mannose residues, forming a class that includes the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN). In addition to recognizing and binding carbohydrates, MMR binds sulfated hormones, including lutropin (luteinizing hormone) and thyrotropin (thyroid-stimulating hormone) through its cysteine-rich domains, and binds serum glycoproteins with accessible mannose and GlcNAc residues. Meanwhile, DC-SIGN binds ICAM-3 and stabilizes DC-T cell interactions.
Of several mannose receptors, MMR is the best characterized and is found in high abundance on dendritic cells and macrophages. MMR recognizes terminal mannose residues on the surfaces of many pathogens and microbial products. In-depth sequence analysis of the intracellular domain of MMR reveals endocytic motifs enabling it to use clathrin-coated pits, which are structures on some kinds of eukaryotic cells that are involved in receptor-mediated endocytosis. After binding to a mannosylated ligand such as MP, MMR quickly directs its molecular cargo toward specialized intracellular compartments in which antigens are processed for subsequent presentation.Recycling motifs on MMR allow it to release such bound molecules and move back to the plasma membrane for successive rounds of antigen capture (Fig. 2).
Cryptococcal MPs Home to and Bind Antigen-Presenting Cells
After characterizing cryptococcal MPs, we tested whether lectin receptors on mammalian cells, including MMR and DC-SIGN, directly interact with and capture MP. We found that Chinese hamster ovary (CHO) cells engineered to express MMR can bind cryptococcal MP. Moreover, the uptake of fluorescently labeled MP by APCs from mice depends on those cells having lectin receptors, and can be inhibited competitively by molecules containing exposed mannose residues. When visualized by microscopy, MP rapidly enters cells through endosomes and subsequently appears to move through the degradative antigen-processing pathway.
We also tested whether MP-binding lectin receptors activate T cells. When APCs take up, process, and present MP to T cells in culture, those T cells become activated, proliferate, and also secrete factors, such as interleukin-2. Tar-geting antigens into APCs via lectin receptors optimizes T cell-stimulation, whereas blocking lectin receptors with competitive inhibitors, or deglycosylating MP, substantially suppresses that stimulation. This T-cell response thus is critically linked to initial recognition of MP by lectin receptors (Fig. 3).
It may be possible to exploit this naturally occurring targeting system to develop vaccines against other diseases. Most licensed vaccines confer protection by virtue of B-cell stimulation and production of high-affinity antibodies. However, T cells are critical for effective defenses against many infectious and neoplastic diseases. Eliciting strong antibody responses is unlikely to completely prevent or treat these diseases. Efforts are currently focused on developing mannosylated vaccine candidates, including ones based on cryptococcal mannoproteins, that promote uptake by DC and subsequent activation of cell-mediated immunity.
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