Meet sea lamprey, the direct descendants of primitive jawless vertebrates from 500 million years ago. Over the last few decades, their immune systems have helped researchers elucidate the adaptive immune responses of ancient sea animals, marking a huge milestone in understanding evolution. Thanks to the adaptive nature of their immune system, sea lampreys can combat pathogens with amazing success rates, even during the first encounter.
Both jawed vertebrates (including us) and sea lamprey contain lymphocytes that respond to pathogens. Whereas the lymphocytes in jawed vertebrates recruit antibodies and T cells to attack pathogens, the ones in sea lamprey follow a different route. Specifically, sea lampreys express a family of proteins called variable lymphocyte receptors (VLRs). Upon introduction of an immunogen, the cells containing VLRs differentiate into plasma cells that secrete VLRB proteins specific to that immunogen.
This mechanism can be leveraged to produce antibodies which are otherwise challenging to produce in conventional immunization methods. Anti-glycan antibody production is one of the fields that can reap tremendous benefits from sea lamprey research.
Why do we even need sea lampreys if there is already substantial research on lectins and monoclonal antibodies for detecting carbohydrate expression? As invaluable as they are in binding to general glycan determinants, lectins have broader specificities for N-glycans and O-glycans (1). Monosaccharides are not unique to either N-glycans or O-glycans, and lectins have weak affinities for monosaccharides. This could cause cross-reactivity when only N-glycans or O-glycans need to be detected. Then we have monoclonal antibodies (mAbs) produced from rodents through immunization with human glycans. However, because humans and rodents have very similar glycomes, immunization often produces weak antibodies bearing non-specific glycan recognition patterns (2).
In contrast, lampreys and mammals have an evolutionary distance of approximately 550 million years. Thus, the VLRBs generated from their immunization could give rise to antibodies that can detect distinct carbohydrate human antigens with higher accuracy, as proven in several studies (3–6).
Researchers from Harvard Medical School, Emory University School of Medicine, and the University of Copenhagen collaborated to exploit the anti-glycan responses of sea lamprey (7). They first immunized lampreys with a set of cell lines comprising distinct glycomes and screened the lamprey plasma against glycan libraries. Then they used yeast surface display (YSD) libraries to clone and enrich VLRB complementary DNA (cDNA) libraries for anti-glycan antibodies, which they termed smart anti-glycan reagents. These reagents could identify distinct glycan epitopes and their cellular locations. Compared to conventional mouse mAbs, the VLRBs produced in sea lamprey displayed superior specificity and affinity.
Immunizing Sea Lamprey with Mammalian Cell Lines
The VLRB production began with immunization with two wild-type cells, two mutant CHO cell lines, pig lung tissue, and human Tn4 cell lines. Then sea lamprey plasma samples were screened on a glycan microarray of 600 unique structures. In every plasma sample, the anti-glycan VLRB profile (i.e., the glycan epitopes they recognized) corresponded to the unique glycan expression profiles of the respective cell lines.
Comparing Lamprey and Mouse Antibodies Response to Antigens
While mAb generation from mice is the most widely accepted method in antibody development, it has several limitations. In many cases, the mice cell lines can tolerate the immunogens from human cells because of glycome similarities, so they fail to generate the desired antibodies.
To demonstrate the superior antibody development capacity of the sea lamprey, the researchers immunized lamprey and mice with human AB blood cells and simian immunodeficiency virus. Although mice did produce anti-glycan antibodies, many of them were already present in mice serum by default; it was difficult to deduce which antibodies were the result of immunization. In contrast, lamprey-derived anti-glycan VLRBs bound a much larger variety of distinct glycan moieties with unique configurations, which the antibodies in mouse serum failed to recognize.
Advantages of Lamprey: Specific, Time and Cost-effective
There are significant advantages of sea lamprey over rodents as anti-glycan antibody resources. The genetic distance to humans is only one example.
The study discusses how VLRBs can improve experimental design feasibility. For example, VLRBs contain a single peptide chain, unlike mouse mAbs with large polypeptide chains.
How does this benefit the use of VLRBs for glycan detection? The main advantage occurs during the construction of cDNA libraries for antibodies. For mouse mAbs, the techniques to produce antibody libraries have several limitations, such as the difficulty of enrichment and loss of specificity. These limitations stem from the dimeric nature of mouse immunoglobulin proteins and the random heavy and light chain pairing. Ultimately, amplifying these proteins requires multiplexing with at least 50 primers, which is costly and time-consuming (8).
In contrast, being single gene products, lamprey VLRBs can be PCR-amplified with a single set of primers. Because of their small modular domains, VLRBs can be incorporated into other proteins without losing their specificity. Lastly, because of their crescent-shaped β-sheet-type binding surfaces, VLRBs recognize binding epitopes that would go unnoticed by Ig-based antibodies with flat binding sites (9).
The Use of YSD Libraries: Discerning Between H-Antigen Presentations
The advantages listed in the previous section enabled the researchers to clone VLRB cDNA libraries onto yeast strains. Pairing the resulting YSD libraries to glycan microarrays, they managed to enrich these libraries for antibodies recognizing a set of glycans with a conserved motif. The 12 VLRB mAbs from 4 immune libraries were designed to detect the type-2 H-trisaccharide motif found in various blood glycans.
Then the researchers compared the specificity of VLRB's from YSD libraries to that of the plant lectin Ulex europaeus agglutinin (UEA-I), conventionally used for H-antigen detection in blood. The binding profiles of the 12 VLRBs revealed that they were each bound to a different type-2 H-antigen presentation. Although all H-antigen structures contained the same binding motif, each VLRB recognized a distinct antigen presentation depending on the glycan structures they carried (e.g., linear vs. branched, N-glycans, O-glycans). Their recognition profiles were similar to 3 VLRBs with previously reported glycan specificity (10). In addition, they did not cross-react with other motifs, such as type 1 and type 3.
The performance of UEA-I resulted in broad specificity, as the researchers reported that it was bound to different H-antigen structures with similar affinity. It was interesting to note non-blood group H antigens among these structures. These results proved how VLRBs could distinguish between minor differences in glycan presentations. Furthermore, the use of YSD libraries enhance the potential of sea lamprey, since YSD libraries are renewable antibody development platforms compatible with several enrichment assays.
VLRBs and Sialic Acid Linkage
The sialylation of glycans is a significant cell biomarker to detect healthy or diseased states. That’s why the researchers also wanted to explore the capability of VLRBs to differentiate between sialylated glycans.
From the H-antigen detection experiments described in the previous section, they identified 3 VLRBs bound to glycans with different sialic acid linkages. The subject of comparison was another plant lectin, this time Sambucus nigra agglutinin (SNA) from Vector Laboratories, a commonly used sialic acid detection reagent.
The binding profiles displayed a clear picture of the affinity of different VLRBs to sialic acid linkages. The 3 VLRBs showed clear peaks for certain N-glycans and O-glycans, with clear preferences towards different sialylated core structures. On the other hand, SNA was broader in its binding profile, lacking any substantial peaks, meaning it bound several sialylated glycans with similar affinity.
For further investigation of distinct binding profiles, the researchers probed 4 types of human tissue arrays (colon, thyroid, lung, and prostate) with the 3 VLRBs and SNA and looked at the staining patterns. While SNA showed similar staining patterns in different tissue arrays, the VLRBs exhibited different stains across the 4 tissue arrays with different sialylated glycan profiles.
Taken together, VLRBs had advantages against the plant lectin SNA by demonstrating preferential binding to certain sialic acid linkages. This also translated successfully to immunohistochemistry applications since they exhibited distinct staining patterns for different tissues.
Challenges for Future Studies
The immunization of sea lampreys gives rise to antibodies that have advantages to mouse mAbs and commercial plant lectins in detecting distinct glycan profiles. These antibodies also prove advantageous in cost and experimental design, such as with the ease of cloning in YSD libraries.
That said, this is not where the story ends, and there is plenty of room for further research. First of all, we still need to find out whether sea lamprey could show self-tolerance to some human antigens, which would lead to weak binding reagents. To elicit self-tolerance trends in sea lamprey, one needs to screen the immunized lamprey plasma against as many glycans as possible.
While large glycan microarrays are available, they only cover a fraction of the vast glycan diversity in the human glycome. In addition, they mostly contain synthetic glycans, meaning some glycan presentations might not even be biologically relevant. In other words, just because a VLRB detects a distinct glycan structure in a microarray does not necessarily mean it will show the same behavior in cell culture or tissue samples. To eliminate the false positives and translate the success of VLRBs into cell biology, one needs to derive glycan microarrays from natural cellular glycomes by releasing glycans from biological samples (11). This advancement will make the results of glycan array screenings more applicable to biomedical research.
- Manimala JC, et al. 2006. High‐Throughput Carbohydrate Microarray Analysis of 24 Lectins. Angewandte Chemie. https://pubmed.ncbi.nlm.nih.gov/16639753/
- Manimala JC, et al. 2007. High-Throughput Carbohydrate Microarray Profiling of 27 Antibodies Demonstrates Widespread Specificity Problems. Glycobiology. https://pubmed.ncbi.nlm.nih.gov/17483136/
- Herrin BR, et al. 2008. Structure and Specificity of Lamprey Monoclonal Antibodies. Proceedings of the National Academy of Sciences of the United States of America. https://pubmed.ncbi.nlm.nih.gov/18238899/
- Luo M, et al. 2013. Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor. Journal of Biological Chemistry. https://pubmed.ncbi.nlm.nih.gov/23782692/
- Nakahara H, et al. 2013. Chronic Lymphocytic Leukemia Monitoring with a Lamprey Idiotope-Specific Antibody. Cancer Immunology Research. https://pubmed.ncbi.nlm.nih.gov/24432304/
- Velásquez AC, et al. 2017. Leucine-Rich-Repeat-Containing Variable Lymphocyte Receptors as Modules to Target Plant-Expressed Proteins. Plant Methods. https://pubmed.ncbi.nlm.nih.gov/28428809/
- McKitrick TR, et al. 2020. Development of Smart Anti-Glycan Reagents Using Immunized Lampreys. Communications Biology. https://www.nature.com/articles/s42003-020-0819-2
- Chao G, et al. 2006. Isolating and Engineering Human Antibodies Using Yeast Surface Display. Nature Protocols. https://pubmed.ncbi.nlm.nih.gov/17406305/
- Boehm T, et al. 2018. Evolution of Alternative Adaptive Immune Systems in Vertebrates. Annual Review of Immunology. https://pubmed.ncbi.nlm.nih.gov/29144837/
- Collins BC, et al. 2017. Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates. Structure. https://pubmed.ncbi.nlm.nih.gov/28988747/
- Song X, et al. 2016. Oxidative Release of Natural Glycans for Functional Glycomics. Nature Methods. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887297/