The story of the cockroach milk protein started about a decade ago with the ‘curiosity of discovering’ and continues with the ‘curiosity of understanding and developing’. While observing these minute crystals inside the embryos of pregnant cockroach females, little did Nathan or Prof. Ramaswamy know that one day it will be popularly known for being the future protein supplement. During these years, while all the authors of the paper collaborated towards the understanding of the protein, we all marveled at the Biology of these cockroaches and the fascinating crystallographic features that the milk proteins exhibited.
Image: Diploptera punctata cockroach (source: livescience.com)
Generally, As an advantage for the organisms, the proteins are under negative natural selection pressure for crystallizing inside their cells. However, the knowledge of several proteins crystallizing inside an organism (in vivo, either in cellulo or ex cellulo) is also known. These proteins are proposed to be under positive selection pressure for crystallizing in vivo with functional importance. The crystals observed by us in the Pacific Beetle cockroaches, usually found near Hawaiian regions, are one of the examples of in vivo crystals. Cockroaches are known to be very sturdy organisms having survived for over 300 million years. During this period, one of the features that it has evolved for higher survival chances is its nature of reproduction. There are three types of reproduction found in the cockroaches; oviparous (eggs-laying), ovo-viviparous (fertilized eggs laid in maternal brood sac without her nourishment) and viviparous (fertilized eggs with maternal brood sac protection and nourishment). The pacific beetle cockroach, scientifically known as Diploptera punctata, is the only known viviparous cockroach till date which gives birth to the young ones like mammals and provides nourishment to the developing embryos. In-keeping with the nomenclature of “milk” used for the maternal nutrition in new-born of mammals, the nourishment provided to the embryos in these cockroaches is also termed the same. The brood sac of the pregnant females secretes this ‘cockroach milk’, which is taken up by the embryos. As the embryos continue to drink the milk, there is a surplus of the protein in their gut. This excess amount is stored inside the embryos’ gut in the form of crystals, which maintain equilibrium with the liquid milk in solution that is readily available to be ingested. Storage of food in the form of crystals allows for a high concentration of food to be stored as well as controlled release of nutrients as needed by the embryos.
Image: From the research article jt5013, showing in vivo-grown Lili-Mip crystals from D. punctata. Polarized microscopy reveals birefringent protein crystals enclosed inside the embryo midgut and an enlarged view of the extracted crystals (inset).
In vivo crystallography is one of the new facets of Structural Biology that deals with structure determination of in vivo protein crystals. Apart from naturally occurring crystals, scientists have also engineered a baculovirus based system to induce in vivo crystal formation. Recent advancements in X-ray free electron lasers (XFEL) and serial femtosecond crystallography (SFX) have resulted in the increase of structures from in vivo crystals. One of the major challenges of in vivo crystallography has been the size of the in vivo-grown crystals that are limited by the volume of the cells that are usually of the micro-nanometer range. However, since these crystals were formed in the gut of the embryos, their sizes were not limited by cellular volumes and were comparatively larger. Therefore, single crystal X-ray diffraction was possible but obtaining the phases of the atoms was tricky. The structure was finally solved using sulfur single wavelength anomalous dispersion method. The protein is a lipocalin with β-barrel forming the lipid binding pocket and a single α-helix. Mass spectrometric and crystallographic studies revealed that each crystal was a heterogeneous mixture of not just multiple protein sequences, but also the sugars bound to these proteins in the form of glycosylation and the fatty acids bound at the binding pocket. More than three sequences of similar proteins (85-95% sequence identity) were found in these crystals. Additionally, multiple N-linked glycosylation sites (3-4 sites) with pauci-mannose and high-mannose structures and variable branching were observed. Further, the fatty acid bound at the pocket was found to be either an oleic acid or linoleic acid. With such an extent of heterogeneity, we were amazed to see that the crystals diffracted X-rays to atomic resolutions of 1.2-1.8 Å.
Generally, in macromolecular X-ray crystallography the rate-limiting step is the crystallization of proteins and obtaining good quality crystals. Usually, when we purify and crystallize recombinant proteins in vitro, we make several modifications such that the protein solutions are homogenous and monodisperse. This usually drives the protein away from its native state in which it naturally occurs. Further, proteins with post-translational modifications have been observed to be heterogeneous and mostly polydisperse in physiological conditions. Hence, when we make these proteins in vitro, we tend to remove all possible glycosylation to enable crystallization of the protein. The high-resolution diffraction of the milk protein crystals and its successful structure determination is till date the first structure reported with this amount of heterogeneity. The precise role for heterogeneity optimized for crystallization in a single lattice is currently unclear. Understanding the molecular structure of these in vivo grown milk protein crystals enables us to appreciate the evolution of viviparity in these cockroaches.
Analysis of the calorific value of these crystals shows that it has three-four times more the energy provided by the equivalent masses of cow, buffalo and other mammalian milks. This cockroach milk protein with proteins, sugars and lipids is a complete food for the embryos. The heterogeneity in the protein sequences provides all the essential amino acids to the embryos. The knowledge of the high energy values gave us an idea that if we produce these milk proteins in vitro in yeast, these recombinant proteins could be used for human consumption as a protein supplement. The curiosity still continues and we hope that this wonderful system can be used for multiple innovations in the future.
Below is the table showing comparison of milk calorific values:
| Species | kcal (per 100 g) |
| Lili-Mip | 230-300 |
| Cow | 66 |
| Goat | 60 |
| Sheep | 95 |
| Water Buffalo | 110 |
| Human | 72 |
About the author:
After completing her Ph.D. from Molecular Biophysics Unit, Indian institute of Science in 2014, Sanchari worked in Syngene International Limited for a very short time. Then she joined the Institute of Stem Cell Biology and Regenerative Medicine (Bangalore, India) as a postdoc. Since the time of joining she has been involved with the cockroach milk protein project. The other areas that interest her are in teaching and education. Her hobbies include dancing and cooking.
To know more, read her paper here: jt5013
This work by ClubSciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
