Research Spotlight: Cells Determine Cell Density using a Small Protein Bound to a Unique Tissue-Specific Phospholipid

Posted on November 23, 2021

Tendon Cofactor

Dr. Schwarz provided fantastic detail of his research interests and his relationship with the Avanti team. In this Lipid Leader Research Spotlight, let’s look at the research that sparked the relationship between Richard Schwarz and Avanti’s Research and Development Team!

How are tendons dependent on lipids for proper function and maintenance?

Cell density is known to alter the rates of cell proliferation and the level of cell differentiation. For proper cellular function, a cell needs to have a real-time assessment of its environment. Improper assessment of cellular density has been associated with cancer and the ability of malignant cells to overgrow the monolayer. Normal cells rely on cell density assessment to slowdown growth and induce differentiated function. The exact “vision” mechanism and how cells “see” is a key area of interest in tendon morphogenesis.

Tendon morphogenesis is regulated by cell density in two ways: (1) rate of cellular collagen production and (2) cell number. This is a result of tendon tissue being composed of over 90% type I collagen. The regulation of cell density in tendon tissue causes a growth plate to form, and an even distribution of collagen along the longitudinal axis of fibrils. In the 1990s, Dr. Schwarz showed that this process and cell density sensing relied on a diffusible factor and identified a large gene that might be responsible, highly conserved between chicken and human.

Finding the Diffusible Factor Responsible for Cell Density Sensing in Tendon Tissue

First, they wanted to make sure that the identified gene was in fact the correct gene responsible. They ran a series of experiments tagging chicken cDNA with polyhistidine and a c-myc polypeptide epitope tags and transfecting them into an osteosarcoma human cell line. This resulted in two observed bands via Western Blotting: the larger, full-length protein and a smaller form at 26kD. Another small band was detected outside of the cell at 28kD. After salt concentrations and ultrafiltering, these larger proteins could be converted to smaller proteins, and a cofactor released to the filtrate while leaving the protein cofactor in the retentate.

And this is where the relationship with Avanti and Dr. Schwarz began. The 28 kD smaller band found outside the cell was determined to have either a covalent modification or bonding to the 26kD band found inside the cell. Further tests – treatment with high salt, low salt, and ultrafiltration – led to the answer that the 28 kD band was due to a binding of a cofactor rather than a covalent modification. When the cofactor was separated from the protein factor, the result was the expected 16 kD protein. So, the next question was, “What is the bone cofactor?”

To find the answer to this question, Dr. Schwarz employed degradative enzymes to cleave the cofactor from the protein and give a partial characterization of the cofactor. To test for a glycolipid, the protein factor/cofactor was treated with polysaccharide degradative enzymes. None of these showed a change in the pattern. Concurrently, lipid degrading enzymes were tested. There were three lipid degrading enzymes that caused a significant shift in the Western blot patterns: lipase, phospholipase C, and sphingomyelinase. Lipase caused the most dramatic shift and showed the 16 kD band seen in tendon cells. The results from the lipase enzyme indicated that the cofactor likely had either a phosphoceramide component or a glycerol fatty acid.

After purification, Mass Spectrometry was used to determine the most likely chemical formula of the bone factor/cofactor (C63H123N2O11P). The MS results showed fragment ions consistent with a ceramide phosphate and a liner moiety. With the bone cofactor analyzed, the investigation shifted back to its primary focus of identifying the tendon factor. Results were similar for lipase, phospholipase C, and phospholipase A2 degradation tests. Sphingomyelin, however, was inactive unlike when tested on the bone factor/cofactor. This result indicated that a ceramide phosphate moiety was missing from the tendon cofactor. After purification of the tendon cofactor, it was analyzed via MS and revealed two rather simple peaks at 668.61 and 696.65, separated by 28. The 668 band was the strongest and so, it was chosen for additional studies. Further testing showed ms/ms fragmentation patters corresponding to two long alkyl fatty acids of 18 and 20 carbons. Knowing that this cofactor was cleaved by phospholipase C means that a phosphate likely connected the two chains. Below was the working structure of the tendon cofactor after the previously mentioned series of tests.

Dr. Schwarz contacted Avanti and asked for help synthesizing the lipid in question. Ultimately, Avanti assisted in proposing an alternative structure to the original working model and synthesized both. The alternative proved to be the active lipid. Dr. Schwarz had this to say about the day he received the lipid synthesized by Avanti’s Research and Development team, “It was an exciting day to open my package and actually see the white powder. For 20 years of research this lipid was part of a predicted factor, missing after enzyme treatment, or a band on a piece of graph paper.”

We want to thank Dr. Schwarz for using Avanti to aid in his fascinating research in tendon morphogenesis. We are excited to see the future of this project and your business! To read the full article, click the following HERE!

If you haven’t had a chance to read the full interview with Dr. Schwarz, you don’t want to miss it! Click HERE to read the full interview! And if you are in the same boat as Dr. Schwarz was and need help making a custom lipid, Contact Us today to discuss your project with our team!

Image Credit: Original Research Publication