By: Richard D. Cummings
I am not aware of studies that have directly and quantitatively determined the number of glycoprotein molecules in a cell or the actual number of N-glycan molecules expressed within the glycoproteins in a cell. N-glycans are those linked to asparagine (Asn) residues in the typical sequon -Asn-X-Ser/Thr-, where X cannot be proline (Pro) (1). However, it is a fun calculation to consider, in which we might use known parameters to approximate the number of N-glycans in a cell. Note: Here we are not considering the intracellular O-GlcNAcylated glycoproteins.
For example, the numbers of many glycoproteins in lymphocytes have been determined. In terms of molecules per cell, consider surface IgM ~13,000 (2), CD22 ~25,000 (3), and the most abundant surface glycoprotein CD45 ~150,000 (4) (also see discussion in Ramya, et al (5)). Note: In some cells it has been estimated that CD45 makes up ~10% of total cell surface proteins (6). Interestingly, for human erythrocytes it has been estimated that there might be ~106 molecules of glycophorin per cell, and that glycoprotein is by far the most abundant in these cells (7).
We might use these numbers to make a rough estimate of the total number of glycoproteins and their associated N-glycans in a cell. Assuming that there could be potentially ~10,000 different glycoproteins maximally expressed by a cell, i.e. ~50% of proteins encoded in the human genome (~20,000 genes) may be N-glycosylated (8). Recent studies on the number of total protein molecules in a human cell suggest ~109protein molecules per cell (9), and others suggest that of the total proteins encoded in the human genome, only slightly more than one-half are expressed (10). (In relation to this, for smaller cells, recent studies on yeast suggest an average of 42 million protein molecules per cell (11).) Furthermore, if the average number of molecules for each one is ~10,000, which is probably an overestimate, then there might be a many as ~108molecules of glycoproteins on the surface of a cell.
It has been determined by N-glycoproteomic analyses (12), that on average the majority of cellular glycoproteins contain ~1-2 N-glycans. Thus, assuming an average of 2 N-glycans per glycoprotein, there would be 2x108 N-glycans per cell or 200 million per cell. Since we have probably overestimated the actual number of glycoprotein molecules per cell by ~10-fold, it might be more reasonable to imagine that there are between ~20 to 200 million N-glycans per cell (9).
In regard to lymphocytes it was estimated that they contain at least 109 molecules of sialic acid per cell (13), whereas for erythrocytes there are ~20 million sialic acids per cell. It has been estimated that the concentration of sialic acid on immune cell surfaces is very high, potentially above 100mM (14).
Thus, assuming that ~10% of total sialic acid is in N-glycans (with the remainder in O-glycans and glycosphingolipids), with some suggesting up to ~40% sialic acid in N-glycans (15), and assuming that the average complex N-glycan contains 2-3 sialic acids/per molecule, then that suggests potentially 108 sialic acids in N-glycans/4 sialic acids per the two average N-glycans on a glycoprotein, or 25 million N-glycans per cell. This is near to our other calculations, in line with the possibility of ~20-200 million N-glycans per cell.
For some protozoans the number of glycoproteins per cell have been estimated a bit more closely. For example, in trypanosomes, the variant surface glycoprotein (VSG) is by far the most dominant glycoprotein (GPI anchored) made by the parasite and occurs at about 107 molecules per cell, and these represent about 10% of the total cell protein! (16). Thus, our estimate above of ~108 molecules of glycoproteins on the surface of a cell is probably within the range of believability. Of course, larger cells might have many more glycoproteins on their surface than a smaller cell.
Such calculations are fun, but clearly imprecise and only provide a ballpark range, but let’s go with the range of ~20-200 million N-glycans per human cell. These are cool numbers to consider, and the actual numbers could of course be lower or higher, as they may be considerably different for different cell types. But such numbers are important to consider in our quest to define the nature of glycoprotein expression and will help to give a better insight into the organization, expression, and density of glycoproteins in biological membranes and secretions.
References:
1. Stanley, P., Moremen, K. W., Lewis, N. E., Taniguchi, N., and Aebi, M. (2022) N-Glycans In Essentials of Glycobiology, 4th Ed. Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, et al., eds. Cold Spring Harbor (NY) 103-116
2. Ovnic, M., and Corley, R. B. (1987) Quantitation of cell surface molecules on a differentiating, Ly-1+ B cell lymphoma J Immunol 138, 3075-3082
3. D'Arena, G., Musto, P., Cascavilla, N., Dell'Olio, M., Di Renzo, N., and Carotenuto, M. (2000) Quantitative flow cytometry for the differential diagnosis of leukemic B-cell chronic lymphoproliferative disorders Am J Hematol 64, 275-281
4. Lavabre-Bertrand, T., Duperray, C., Brunet, C., Poncelet, P., Exbrayat, C., Bourquard, P. et al. (1994) Quantification of CD24 and CD45 antigens in parallel allows a precise determination of B-cell maturation stages: relevance for the study of B-cell neoplasias Leukemia 8, 402-408
5. Ramya, T. N., Weerapana, E., Liao, L., Zeng, Y., Tateno, H., Liao, L. et al. (2010) In situ trans ligands of CD22 identified by glycan-protein photocross-linking-enabled proteomics Mol Cell Proteomics 9, 1339-1351
6. Thomas, M. L. (1989) The leukocyte common antigen family Annu Rev Immunol 7, 339-369
7. Tayyab, S., and Qasim, M. (2020) Biochemistry and Roles of Glycophorin A Biochemical Edu 16, 63-66
8. Apweiler, R., Hermjakob, H., and Sharon, N. (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database Biochim Biophys Acta 1473, 4-8
9. Milo, R. (2013) What is the total number of protein molecules per cell volume? A call to rethink some published values Bioessays 35, 1050-1055
10. Djebali, S., Davis, C. A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A. et al. (2012) Landscape of transcription in human cells Nature 489, 101-108
11. Ho, B., Baryshnikova, A., and Brown, G. W. (2018) Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome Cell Syst 6, 192-205 e193
12. Gao, Y., Shen, L., Dong, T., Yang, X., Cui, H., Guo, Y. et al. (2022) An N-glycoproteomic site-mapping analysis reveals glycoprotein alterations in esophageal squamous cell carcinoma J Transl Med 20, 285
13. Kataoka, S., Kikuchi, T., and Toyota, T. (1985) Expression of receptors for Fc portion of IgM (Fc mu . R) and surface neuraminic acid on the human peripheral lymphocytes Tohoku J Exp Med 145, 73-84
14. Collins, B. E., Blixt, O., DeSieno, A. R., Bovin, N., Marth, J. D., and Paulson, J. C. (2004) Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact Proc Natl Acad Sci U S A 101, 6104-6109
15. Torii, T., Yoshimura, T., Narumi, M., Hitoshi, S., Takaki, Y., Tsuji, S. et al. (2014) Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex Glycoconj J 31, 671-683
16. Manna, P. T., Boehm, C., Leung, K. F., Natesan, S. K., and Field, M. C. (2014) Life and times: synthesis, trafficking, and evolution of VSG Trends Parasitol 30, 251-258