A unique covalent bond in basement membrane is a primordial innovation for tissue evolution

Significance

The evolution of multicellular animals from single-celled ancestors was one of the most significant transitions of life on earth. The emergence of larger, more complex animals able to resist predation and colonize new environments was enabled, in part, by a collagen scaffold, which anchors cells together to form tissues and organs. Here, we show that a unique chemical bond, a link between sulfur and nitrogen atoms called a sulfilimine bond, arose over 500 Mya, binding this scaffold together and enabling tissues to withstand mechanical forces. Peroxidasin forms the bond by generating hypohalous acids as strong oxidants, a form of bleach, which normally function as antimicrobial agents. These understandings may lead to approaches for targeting tumors and treatment of other diseases.

Fig. 1.  The sulfilimine bond stabilizes collagen IV scaffolds by the cross-linking of triple helical building block protomers.
(A) The sulfilimine bond cross-links Met93 and Hyl211 at the interface between the trimeric NC1 domains of two adjoining protomers, forming a globular hexamer structure.
(B) Dimeric subunits reflect the presence of the sulfilimine bond in human collagen IV by immunoblot (JK2 Ab) and protein stain.
(C) MS analysis of tryptic peptides derived from dimeric subunits verified the presence of the bond by a mass difference of 2.0299 between theoretical mass of uncross-linked and observed mass of cross-linked peptides and subsequent multistep CID fragmentation (MS2/MS3) analyses.

Fig. 2. Multiple sequence alignment of collagen IV NC1 domains encompassing Met93 and Hyl211 amino acid residues and Pxdn among 11 metazoan and 1 protozoan phyla.
(A) Met93 and Lys/Hyl211 (yellow) are conserved in all eumetazoans, except for the cnidarian H. magnipapillata, and they are absent in the phyla of Placozoa and Porifera and the protozoan phylum Choanozoa. All sequences belong to the collagen IV α1-like subfamily of chains, except for Drosophila (viking) and Ascaris (α2 chain).
(B) Schematic representations of Pxdn. Pxdn sequence was incomplete on both ends for Mytilus, Clytia, Trichoplax, and Monosiga and short on one end for Saccoglossus, which is indicated here by a shortened schematic representation. Sequence data were gathered from *National Center for Biotechnology Information Reference Sequence, †gathered from whole-genome shotgun/transcriptome shotgun assembly, §generated by RNA-Seq analysis of animal tissues, or ¶assembled from cDNA libraries. All National Center for Biotechnology Information GenBank accession numbers are listed in Table S1.

Fig. 3. NC1 hexamers were excised from animal basement membranes and analyzed by SDS/PAGE as shown in Fig. 1 A and B. The dimeric subunits, which indicate the presence of the bond, were found in nine major eumetazoan phyla. Among eight cnidarians investigated, only Hydra NC1 lacked dimeric subunits. All NC1s were immunoblotted against the rat monoclonal antibody, JK2, except for C. elegans (rabbit polyclonal; NW-154) and Drosophila (mouse monoclonal; 6G7). Black outlines indicate the locations of cropping for blot images. 

Fig. 4. Expression of collagen IV and Pxdn during development in zebrafish and morpholino (MO) knockdown of peroxidasin in zebrafish embryos. (A) Pxdn and collagen IV expression during zebrafish embryonic development. Real-time qPCR studies were conducted to examine expression levels of Pxdn, collagen4α1, and collagen4α2. *Student t test P value < 0.03 compared with expression at 1,000 cells. Error bars = SEM. Blue, pxdn; red, col4a; black, col4a2. (B) Control and (C) Pxdn MO groups. MO-injected embryos displayed (D) general severe defects that include cardiac edema, smaller eyes, and gross trunk patterning defects (4/45), (E) partial curved trunk (21/45), or (F) normal development (20/45). (G) SDS/PAGE analysis of Pxdn MO embryonic phenotypes at 24 hpf by Western blot. Collagenase digests were normalized for total protein load by protein stain with SYPRO-Ruby (Fig. S8).


Abstract

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Footnotes

1A.L.F., R.M.V., and S.V.C. contributed equally to this work.

2A list of The Aspirnaut coauthors can be found in Table S2. Aspirnaut is a K--20 Science, Technology, Engineering, and Math (STEM) pipeline program for diversity that partners the experiential and content expertise of Vanderbilt University with rural kindergarten through 12th grade schools and diverse high school, undergraduate, and graduate students.

3To whom correspondence should be addressed. E-mail: billy.hudson@vanderbilt.edu.

Author contributions: R.M.V., S.V.C., V.K.P., V.P.Y., M.T.I., J.K.H., and B.G.H. designed research; A.L.F., S.V.C., G.B., V.P.Y., C.L.S., K.L.R., W.H.M., T.A.C., D.-B.B., R.E.S., and T.A. performed research; G.B. contributed new reagents/analytic tools; A.L.F., R.M.V., S.V.C., V.K.P., V.P.Y., D.-B.B., and R.E.S. analyzed data; and A.L.F. and B.G.H. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. GAMX01000001, GAMX01000002, GAND01000001, GAND01000002, GANB01000001, GANB01000002,GAMY01000001, GAMY01000002, GANA01000001, GANA01000002, GAMZ01000002, and GANC01000002).

This article contains supporting information online at 

www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318499111/-/DCSupplemental.

Freely available online through the PNAS open access option. (Full Text)

ORIGINAL: PNAS

Aaron L. Fidlera,1, 

Roberto M. Vanacorea,b,1, 

Sergei V. Chetyrkina,b,1, 

Vadim K. Pedchenkoa,b,

Gautam Bhavea,b, 

Viravuth P. Yinc, 

Cody L. Stothersa, 

Kristie Lindsey Rosed,e, 

W. Hayes McDonaldd,e,f,

Travis A. Clarkg, 

Dorin-Bogdan Borzaa,b, 

Robert E. Steeleh, 

Michael T. Ivyi, 

The Aspirnautsb,2,

Julie K. Hudsonj, and 

Billy G. Hudsona,b,c,d,f,k,l,3

Author Affiliations

Edited* by Mina J. Bissell, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved November 22, 2013 (received for review September 30, 2013)

The gold standard for cell penetration

ORIGINAL: MIT
David L. Chandler, MIT News Office
August 23, 2013

Gold nanoparticles with special coatings can deliver drugs or biosensors to a cell’s interior without damaging it.

Illustration shows the passage of a gold nanoparticle (in orange) covered with a monolayer of hydrophobic/hydrophilic material (shown in blue-green, yellow and red), passing through a cell membrane composed of lipids (white and blue). Graphic courtesy of Reid Van Lehn

Cells are very good at protecting their precious contents — and as a result, it’s very difficult to penetrate their membrane walls to deliver drugs, nutrients or biosensors without damaging or destroying the cell. One effective way of doing so, discovered in 2008, is to use nanoparticles of pure gold, coated with a thin layer of a special polymer. But nobody knew exactly why this combination worked so well, or how it made it through the cell wall.

Now, researchers at MIT and the Ecole Polytechnique de Lausanne in Switzerland have figured out how the process works, and the limits on the sizes of particles that can be used. Their analysis appears in the journal Nano Letters, in a paper by graduate students Reid Van Lehn, Prabhani Atukorale, Yu-Sang Yang and Randy Carney and professors Alfredo Alexander-Katz, Darrell Irvine and Francesco Stellacci.

Until now, says Van Lehn, the paper’s lead author, “the mechanism was unknown. … In this work, we wanted to simplify the process and understand the forces” that allow gold nanoparticles to penetrate cell walls without permanently damaging the membranes or rupturing the cells. The researchers did so through a combination of lab experiments and computer simulations.

The team demonstrated that the crucial first step in the process is for coated gold nanoparticles to fuse with the lipids — a category of natural fats, waxes and vitamins — that form the cell wall. The scientists also demonstrated an upper limit on the size of such particles that can penetrate the cell wall — a limit that depends on the composition of the particle’s coating.

The coating applied to the gold particles consists of a mix of hydrophobic and hydrophilic components that form a monolayer — a layer just one molecule thick — on the particle’s surface. Any of several different compounds can be used, the researchers explain.

“Cells tend to engulf things on the surface,” says Alexander-Katz, an associate professor of materials science and engineering at MIT, but it’s “very unusual” for materials to cross that membrane into the cell’s interior without causing major damage. Irvine and Stellacci demonstrated in 2008 that monolayer-coated gold nanoparticles could do so; they have since been working to better understand why and how that works.

Since the nanoparticles themselves are completely coated, the fact that they are made of gold doesn’t have any direct effect, except that gold nanoparticles are an easily prepared model system, the researchers say. However, there is some evidence that the gold particles have therapeutic properties, which could be a side benefit.

Gold particles are also very good at capturing X-rays — so if they could be made to penetrate cancer cells, and were then heated by a beam of X-rays, they could destroy those cells from within. “So the fact that it’s gold may be useful,” says Irvine, a professor of materials science and engineering and biological engineering and member of the
Koch Institute for Integrative Cancer Research.

Significantly, the mechanism that allows the nanoparticles to pass through the membrane seems also to seal the opening as soon as the particle has passed. “They would go through without allowing even small molecules to leak through behind them,” Van Lehn says.

Irvine says that his lab is also interested in harnessing this cell-penetrating mechanism as a way of delivering drugs to the cell’s interior, by binding them to the surface coating material. One important step in making that a useful process, he says, is finding ways to allow the nanoparticle coatings to be selective about what types of cells they attach to. “If it’s all cells, that’s not very useful,” he says, but if the coatings can be targeted to a particular cell type that is the target of a drug, that could be a significant benefit.

Another potential application of this work could be in attaching or inserting biosensing molecules on or into certain cells, Van Lehn says. In this way, scientists could detect or monitor specific biochemical markers, such as proteins that indicate the onset or decline of a disease or a metabolic process.

In general, attachment to nanoparticles’ surface coatings could provide a key to cells’ interiors for “molecules that normally wouldn’t have any ability to get through the cell membrane,” Irvine says.

Vince Rotello, a professor of chemistry at the University of Massachusetts at Amherst who was not involved in this research, says this work is “careful, well thought out and elegantly presented.” He adds, “This study provides a very interesting alternative mechanism to cell uptake of nanomaterials that could open up new therapeutic pathways.”

The work was supported by the National Science Foundation, the National Cancer Institute and the U.S. Army Research Office.

Origin of Life: Hypothesis Traces First Protocells Back to Emergence of Cell Membrane Bioenergetics

ORIGINAL: Science Daily

Dec. 20, 2012 — A coherent pathway -- which starts from no more than rocks, water and carbon dioxide and leads to the emergence of the strange bio-energetic properties of living cells -- has been traced for the first time in a major hypothesis paper in Cell this week.

A major new hypothesis outlines a coherent pathway that starts from no more than rocks, water and carbon dioxide and leads to the emergence of the strange bio-energetic properties of living cells. (Credit: iStockphoto/Henrik Jonsson)

At the origin of life the first protocells must have needed a vast amount of energy to drive their metabolism and replication, as enzymes that catalyse very specific reactions were yet to evolve. Most energy flux must have simply dissipated without use.

So where did it all that energy come from on the early Earth, and how did it get focused into driving the organic chemistry required for life?

The answer lies in the chemistry of deep-sea hydrothermal vents. In their paper Nick Lane (UCL, Genetics, Evolution and Environment) and Bill Martin (University of Dusseldorf) address the question of where all this energy came from -- and why all life as we know it conserves energy in the peculiar form of ion gradients across membranes.

"Life is, in effect, a side-reaction of an energy-harnessing reaction. Living organisms require vast amounts of energy to go on living," said Nick Lane.

Humans consume more than a kilogram (more than 700 litres) of oxygen every day, exhaling it as carbon dioxide. The simplest cells, growing from the reaction of hydrogen with carbon dioxide, produce about 40 times as much waste product from their respiration as organic carbon (by mass). In all these cases, the energy derived from respiration is stored in the form of ion gradients over membranes.

This strange trait is as universal to life as the genetic code itself. Lane and Martin show that bacteria capable of growing on no more than hydrogen and carbon dioxide are remarkably similar in the details of their carbon and energy metabolism to the far-from-equilibrium chemistry occurring in a particular type of deep-sea hydrothermal vent, known as alkaline hydrothermal vents.

Based on measured values, they calculate that natural proton gradients, acting across thin semi-conducting iron-sulfur mineral walls, could have driven the assimilation of organic carbon, giving rise to protocells within the microporous labyrinth of these vents.

They go on to demonstrate that such protocells are limited by their own permeability, which ultimately forced them to transduce natural proton gradients into biochemical sodium gradients, at no net energetic cost, using a simple Na+/H+ transporter. Their hypothesis predicts a core set of proteins required for early energy conservation, and explains the puzzling promiscuity of respiratory proteins for both protons and sodium ions.

These considerations could also explain the deep divergence between bacteria and archaea (single celled microorganisms) . For the first time, says Lane, "It is possible to trace a coherent pathway leading from no more than rocks, water and carbon dioxide to the strange bioenergetic properties of all cells living today."

Writing Messages With Water

ORIGINAL: Tech News Daily

By Julian Taub, 

December 04 2012 05:53 PM ET

Examples of hydroglyphic writing. 

CREDIT: Wyss Institute for Biologically Inspired Engineering

Scientists have used nanotechnology to create “selectively wet” materials that can be used to write long-lasting messages with water.

The concept, called "hydroglyphics," was exhibited by scientists at Harvard who recently teamed up with a group of Merrimack, N.H., high school students and faculty to make an educational demo. 

The demo, appropriately entitled "Hydroglyphics," helps people visualize the difference between water repelling and wetting surfaces. The main principle behind hydroglyphics (a combination of the words “hydro” and “hieroglyphics”) is that by changing the properties of a surface, you can make your own special prints using water. All you need is some foam stickers, a modified Tesla coil and a Petri dish.

[VIDEO: Nanotechnology's Big Impact]

Each audience member takes a Petri dish and chooses a favorite sticker, tacking it onto the bottom of the dish. The demo performer then puts each dish under the Tesla coil, and zaps them. A purple spark appears accompanied by a loud noise. Once the sticker is removed, water is added to the dish. The water fills up everywhere except on the area where the sticker had been, creating an “engraving.” The message can last about one month.

Harvard scientist Philseok Kim, the first author on the paper about this demo, stumbled upon the idea for hydroglyphics. While helping one of Merrimack’s teachers, Raymond Sleeper, come up with a new demo, he experimented with equipment in the lab that was being used for other research projects.

“I tested [the demo on] a Petri dish, to see how much contrast between hydrophilicity and hydrophobicity we could make,” Kim, of the Wyss Institute, said. “To our surprise, it nicely generated strikingly good contrast.”

The Petri dishes originally have a plastic structure that repels water (hydrophobic). When treated with the Tesla coil, the air becomes conductive and oxygen combines with the plastic, making the Petri dish surface attracted to water (hydrophilic). However, the area under the sticker was protected from the air, so it still repels water. Water in the dish sticks to the hydrophobic regions, keeping the message area dry. 

The hydroglyphics demo has been a success wherever the researchers have taken it, Kim said. “Purple electric arcs with funny zapping noise, cute and colorful stickers, and mystery messages… all of them are a series of 'wow' moments that just happen over a few minutes. I think this is a truly engaging combination.”

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NBDNano doing a conceptual design of a self-filling water bottle

ORIGINAL: NDBNano

NBD makes use of a nano-scale surface to enhance water condensation. Mimicking the Namib Desert Beetle, our nanotechnology can be used to collect water in the most arid regions of the world. 

Our Technology

(Photo From: Moongateclimber/Wikipedia Commons )

Superhydrophobic and superhydrophilic surfaces have a variety of commercial applications. NBD is targeting water harvesting by employing these alternating chemical surface features in varied micro-patterns/arrays affording unique and uncharacteristic properties. Our surface engineering solutions are based on years of research of coming from elite university laboratories in a range of fields, including materials science and chemical engineering.

Applications:

• Enhanced dehumidification for house-hold consumers
• Production of potable water for military operations
• Production of water for greenhouses to support plant life
• Production of potable water for third world nation

NBD is currently in a heavy R&D phase, during which we will be optimizing our chemical coatings and building prototypes of devices to utilize these coatings - including a conceptual design of a self-filling water bottle. We will also be exploring additional uses of these unique coatings, in order to best position ourselves for a launch in 2014-2015. Thank you for your support!

Our Team

Miguel

Miguel is a co-founder of NBD with a background in startups. While this is his second stint at starting a company, he also has worked at TechStars, an early stage investor in seed startups. He has a degree in Biology from Boston College.

Andy

Andy is a co-founder and lead chemist. Andy has several years of experience in organic chemistry and has presented his work at a number of conferences including local, national, and international venues. Andy is now a grad student at MIT working towards his PhD in Organic Chemistry. He is a graduate of Trinity College.

Deckard

Deckard is a co-founder of NBD with a background in biomimicry. Deckard had the inspiration for this company while taking a class about the Namib Desert Beetle. He has a degree in Biology with a concentration in bioinformatics from Boston College.

Rob 

Rob is the lead engineer for NBD. Rob is a mechanical engineer from Northeastern University and has a widespread background in aerospace, medical, and consumer product design. Rob has also worked as a design consultant for several Boston based startups.

Nobel de Química 2012: Robert J. Lefkowitz y Brian K. Kobilka

ORIGINAL: La Vanguardia

Josep Corbella. Barcelona

10/10/2012

La Real Academia de Ciencias de Suecia ha concedido el galardón a los científicos estadounidenses Robert Lefkowitz y Brian Kobilka por sus investigaciones sobre un tipo de receptores de la membrana de las células que regulan múltiples funciones biológicas

Los científicos estadounidenses Robert J. Lefkowitz y Brian K. Kobilka han sido galardonados con el Premio Nobel de Química 2012 AP / EFE / Archivo

Los científicos estadounidenses Brian Kobilka y Robert Lefkowitz han ganado el premio Nobel de Química 2012 por sus investigaciones sobre un tipo de receptores de la membrana de las células que regulan múltiples funciones biológicas.

De los receptores acoplados a proteínas G, como se denominan, depende la actividad de hormonas como la adrenalina o la leptina, así como de neurotransmisores como la serotonina o la dopamina. Regulan, por lo tanto, desde el apetito al estado de ánimo, pasando por la tensión arterial, el tono muscular o las reacciones ante situaciones de estrés.

Aproximadamente la mitad de los fármacos existentes actualmente basan su eficacia en la acción de estos receptores, ha destacado la Real Academia de Ciencias de Suecia al anunciar el galardón. Su conocimiento detallado, gracias a las investigaciones de Kobilka y Lefkowitz ayudará a desarrollar nuevos fármacos más eficaces y con menos efectos secundarios.

Kobilka (Little Falls, Minnesota,1955) es profesor de la Universidad de Stanford en California. Lefkowitz (Nueva Yordk, 1943) es profesor de la Universidad Duke en Durham (Carolina del Norte). Ambos compartirán los 8 millones de coronas suecas (unos 900.000 euros) del premio.

La presente edición de los Premios Nobel arrancó el lunes con la concesión al británico John B. Gurdon y al nipón Shinya Yamanaka del de Medicina, y prosiguió ayer con el anuncio de que el Nobel de Física recayó en el francés Serge Haroche y el estadounidense David J. Wineland.

En los próximos días, la Academia Sueca seguirá dando a conocer el nombre de los ganadores de los Premios Nobel por este orden: mañana jueves 11 será el turno del Nobel de Literatura, seguido por el Nobel de la paz, el viernes día 12.

Por último, el próximo 15 se conocerá el ganador del premio Nobel de Economía. El de Economía, por cierto, es el único de los galardones que no quedó estableció por Alfred Nobel en su testamento, sino que se incorporó a la lista en 1969.