The skin vitally protects the body of all mammalians. This protection is created by the epidermis, which is composed by layers of skin cells called keratinocytes. These keratinocytes tightly attach to one another by structures call desmosomes.
At desmosomes, keratinocytes attach to one another via molecules called desmocollin and desmoglein. The completion of The Human Genome Project in April 2003 gave us the ability to read nature's complete genetic blueprint for building a human being and know that, indeed, human have only 6 desmocollins (Dsc1a, 1b, 2a, 2b, 3a, and 3b) and 4 desmogleins (DSG1, DSG2, DSG3, and DSG4).
Dr. Nguyen is the first scientist who discovered desmoglein 4 (DSG4) molecule. He was the first who identified the evidence of desmoglein 4 protein and cloned the molecule's mRNA when he studied a skin disease called pemphigus vulgaris (PV). It is a potential lethal disease in which patients develop antibodies against their own skin cells that make them detach from one another and make big skin blisters (FIGURE 1). The symptom of this disease can be controlled by drugs such as glucocorticosteroids, but the disease still cannot be cured. Dr. Nguyen wants to know what really cause the disease.
It was believed by some scientists that the disease is caused by antibodies against desmoglein 3 (DSG3) alone because many patients have antibodies against this molecule and, when scientists used antibodies against DSG3 to inject into neonatal mice, an animal model for the disease, the animal developed skin blisters like patients. Many scientific evidences suggest that cellular signal trasduction is critical for the pathomechanism of the the disease. But structurally DSG3 does not have the concensus segment that is needed for signalling function, suggesting a very similar molecule that structurally has signaling segment could exist and be targeted by pathogenic autoantibodies in pemphigus vulgaris patients. These authors suggested that autoantibodies to both desmoglein 1 (DSG 1) and DSG3 are necessarily to produce the skin lesions in PV (DSG1 molecule has the consensus signaling segment). But they also suggested that autoantibody to DSG3 alone can cause the lesion in patients' mucosa.
When Dr. Nguyen did the same passive transfer experiment of PV autoantibodies that did not have anti-DSG1 to the genetic mice that did not have DSG3 molecule, the mice also developed skin blisters. Pure and simple, he proposed that there was a new target molecule for the disease-causing antibodies and the potential novel molecule could be very similar to DSG3.
First evidence of DSG4: In 1999, he use a technique, called Western Blot, of proteins isolated from genetic mice lacking of desmoglein 3 and use affinity purified antibodies against DSG3 protein as the probe, he found the first evident of desmoglein 4 (DSG4) protein. The molecule indeed has the size about 130-140 Kd (similar to DSG 3). (Nguyen et al, Journal of Clinical Investigation. 2000 (12):1467-79.)
The next step Dr. Nguyen did was to identify the actual identification of this novel molecule. He was one of the first scientists who have used the results of The Human Genome Project when it has not been completed to discover new molecules.
The Human Genome Project - one of the great feats of exploration in history-is an international research effort to sequence and map all the genome of human. The project completed in April 2003, however, much earlier than that, since early 2002, a large amount of data of human genome from this project had been submitted to the GenBank and therefore available to the public. These data also include most of the DNA sequence of human chromosome 18.
With these data in hand, to discover a novel molecule and its transcript mRNA sequence, unlike in the old time, today scientists can do a short cut by starting with a computer and some gene predicting softwares to predict the possible mRNA sequence of a new genes.
Indeed, since early 2002, using some computer softwares such as Genescan and Twinscan, the NCBI Annotation Project (National Center for Biotechnology Information, USA) predicted several potential novel genes such as cadherin 22, 23, and 24 etc, and submitted them to Genbank that available to the public. The sequences that were predicted by these softwares at that time, however, could be inaccurate or predict the genes that could even actually did not exist.
With the predicted sequence, scientists could prove if the molecule actually exists by simple laboratory experiments such as reverse transcribed polymerase chain reaction (RT-PCR) that is very efficient with the specific and unique sequence of each potential novel molecule.
Since the computer predicted sequence can be wrong, a claim of a novel molecule is only valid if the author(s) can provide actual laboratory data such as the photography of the experimental results and the real critical sample of sequencing electropherogram that shows that they actually did the experiment; and most importantly, the data must be reproducible by independent scientists resulting an identical mRNA sequence of the novel molecule.
Use silico-cloning to predict an existence of DSG4: In early 2002, Dr. Nguyen used his novel technique that he called “Silico cloning” (or cloning by computer, that he believed that many other scientists could easily come up the same idea) to find a possible novel molecule that was similar to DSG3 . “Almost every evening, I had dinner in front of my computer, entered the NCBI website, and used a tool called Basic Local Allignmeent Search Tool (BLAST) to search for any new DNA sequence that appeared to be similar to desmoglein 3 and align them logically looking for novel molecule…” he said.
He found several transcribable DNA segments similar to DSG3 gene, which he aligned them to construct a computational predicted sequence that was very similar to DSG3. Using the BLAST tool to the genome sequence, he knew that it was predicted from the DNA sequence between the locations of DSG3 and DSG1 genes in chromosome 18q12.1. He called this potential novel molecule desmoglein 4 (DSG4). The predicted mRNA molecule encode for a protein about the size of DSG1 (about 160 kDa).
Use RT-PCR to clone the actual DSG4 mRNA, not simple as expected: Dr. Nguyen then used RT-PCR to clone DSG4 but at first he was able to clone only two third of the molecule at the 5’ end. It appeared that that the computational predicted Desmoglein 4 sequence was not correct at the 3’ end region therefore the primers designed from the data based on computational-predicted DSG4 sequence did not give any results. Thus the portion of the predicted molecule that was used to design primers for RT-PCR could belong to an intron. Therefore the actual molecule should be smaller than 160 kDa. This was the most challenging part to get the correct complete DSG4 mRNA and it took Dr. Nguyen over 10 months.
Then he down loaded the whole chromosomal DNA sequence of this region as well as some other small EST clones that had sequences similar to DSG3 to design new primers for RT-PCR to find the remained actual portion of DSG4 (FIGURES 2 & 3).
By sequencing the RT-PCR products (FIGURE 3), Dr. Nguyen identified the actual sequence of human DSG 4. The resulted sequences constructed a 3531bp cDNA containing a complete open reading frame of 3180 nucleotides encoding for a deducted 1059 amino acid polypeptide with predicted molecular weight of 115.5 kDa for non-glycosylated molecule. Removing of the leading peptide from the site for proteolytic cleavage (RRQKR) predicts a mature non-glycosylated molecule with MW of 109.5kDa (which is similar to MW of non-glycosylated DSG3, 107.5kDa, that explains why the two molecules co-migrate at the same position at about 130 kDa in electrophoresis gel).
He submitted the sequence to the GenBank on October 25, 2002 and his record was assigned the accession number AY168788.1. It is the first laboratory complete cloned DSG4 mRNA sequence has ever been published (and it should have been listed in the first publication of DSG4 that Dr. Nguyen and his colleaguage pusblished on journal Cell in March 2003 beside his book chapter on pemphigus that was published by CRC in 2003). It appeared that the actual sequence of human DSG4 is significantly different from the computational-predicted sequence in the region at 3’ end, which is the portion of the molecule that carries the conserved signaling sequence. It differs of 117 bases that transcibable to 39 amino acids, actually belong to an intron. RT-PCR using primers that were designed based on the different region of the computational-predicted DSG4 mRNA sequence did not give any result.
Actual size of DSG4 molecule: About a month after Dr. Nguyen reported the actual DSG4 mRNA sequence, in November 2002, other researchers reported DSG4 mRNA that were identical to the computer predicted molecule (encoding a 160 kDa protein), which was assigned as DSG4 variant 2. The publication of this report in JID in January 2023 show no actual laboratory evidence of any RT-PCR done. This computer predicted data led these workers and others from somewhere else disputed the role of DSG4 in PV, arguing that DSG4 was 160 kDa, not 130 kDa.
On January 31, 2007, a group of 83 scientists lead by Dr. Strausberg at the National Cancer Institute, Bethessda, MD, USA submitted to the GenBank their desmoglein 4 mRNA sequence (GenBank: BC132907.1). Their sequence confirms 100% accuracy of the sequence that Dr. Nguyen reported in October 2002.
The desmoglein 4 mRNA sequence is now has an NCBI Reference Sequence: NM_001134453.1. It is called Homo sapiens desmoglein 4 (DSG4), transcript variant 1, mRNA. In this reference, base 107-3637 that encodes complete DSG4 protein is 100% identical to bases 1-3531 of the sequence from GenBank: AY168788.1. (FIGURE 4). And of course, the protein sequence is 100% identical to the DSG4 protein sequence reported by Dr. Nguyen on October 25, 2002 (FIGURE 5).
Over the years since the DSG4 mRNA coding for the 130 kDa protein of 1059 amino acid was reported by Dr. Nguyen and the bigger "variant 2" of the 160 kDa DSG4 reported by others, the anotation project of NIH of USA has corrected and now we see only the smaller "variant 2" with mRNA coding for 1040 amino acid and the sequence are virtually identical to the first DSG4. Thus there is only one DSG4.
With regard to the actual size of DSG4 protein, over the years, many biotech companies have developed mouse or rabit antibodies specific for DSG4 that do not cross react with either DSG3, DSG2, or DSG1, these antibodies specifically reconized a 130 kDa protein on Western blot of human epidermal extract. One example, the MAB6589 human Desmoglein-4 monoclonal antibody produced by R&D Systems (www.rndsystems.com, FIGURE 6). Thus there is no such a 160 kDa DSG4.
Location of DSG4 gene in human genome: With the correct mRNA sequence of the novel desmoglein 4 (DSG4), Dr. Nguyen used the BLAST tool to define the exact 15 exons and 14 introns of desmoglein 4 gene and their locations in the chromosome 18q12.1. The result is different from the computational-predicted DSG4 sequence that showed 16 exons and 15 introns (FIGURE 7).
The order of arrangement of desmoglein cluster genes, DSG2 – DSG3 – DSG4 – DSG1 in chromosome 18 is probably not a coincidence but rather a result of evolution. It most likely represents the order of protein expression according to the differentiating levels of skin cells (keratinocytes); i.e; DSG2 expresses at the lowest layer of the epidermis, DSG3 and DSG4 express at higher keratinocyte layers, while DSG1 expresses at highest/most differentiated keratinocyte layers (FIGURE 8).
Expression of DSG4 in epidermis: It is not surprised that there are some differences in the laboratory data of the DSG4 protein expression in skin found in different publications. The DSG4 publication on journal Cell shows that DSG4 express all over the suprabasal keratinocyte layers of the epidermis; while those in some other publications showed only the expression of DSG4 on the top-most differentiated layer. The reason is because different techniques were used in these publications:
RNA In-situ hybridization (hybridization histochemistry) technique uses labeled oligonucleotides specific for DSG4 mRNA sequence therefore it stains any cells where DSG4 mRNA expresses. When the technique was done in parallel with indirect immunofluorescence using DSG4 polyclonal antibody, the result is very reliable (results from DSG4 publication on journal Cell 2003; 113(2): 249-60).
The DSG4 polyclonal antibody is very specific and sensitive to the DSG4 protein in natural form because they were generated by immunizing animal with a large portion of DSG4 that would fold more correctly in the immunized animals. In addition, the antibodies recognize several epitopes at different presentation therefore very sensitive (results from DSG4 publication on journal Cell 2003; 113(2): 249-60).
On the other hand, DSG4 monoclonal antibody could be very specific but less sensitive to the DSG4 protein in natural form. They were usually generated by immunizing animals with a small peptide (only few amino acids - epitope) of DSG4 that was linked to synthetic carriers. Therefore the presentation of the complex in the immunized animals could not be the same as natural DSG4 protein. In addition, even in the case a large portion of the molecule was used for immunization, the monoclonal antibody is generated by a single clone of antibody making cells, it can recognize only one epitope presented in one specific presentation, thus much less sensitive to the targeted protein, and usually more sensitive with the protein in the degraded or denature form, ie., in Western blot or in the stratum cornium of the epidermis where a keratinocyte reach to the end of its life - cell death, its protein degraded.
Some one also tried to use electron microscope to "define" the location of DSG4 in epidermis. This is not a typical technique for this purpose. Simply said, if we want to see the where in the field the flower grow, we look the whole field to see the full picture, not only narrow our eyes to one spot of the flowers without the ability to the whole field.
Since the expression of DSG4 protein in the epidermis is dynamic according to the level of cell differentiation, only at certain presentation where the molecule can expose the epitope that is presented the same specific way as if it was presented by the carrier in immunized animal, it could be recognized by DSG4 monoclonal antibodies (results from DSG4 publication in other journals). In summary, the finding of DSG4 expression through out the suprabasal epidermis presented in DSG4 publication in journal Cell in 2003 could be the most accurate one by far (FIGURE 8).
Dr. Nguyen also analyzed the sequence of desmoglein 4 and found several interesting features. Overall, DSG4 shares 79%, 68% and 48% similarity to DSG3, DSG2, and DSG1 respectively. But at the N-terminal regions of these molecules, DSG4 shares a much higher level of similarity to DSG3 and DSG1: 89% and 92% respectively. Along these similar extracellular domains of the molecules, there are several identical amino acid stretches that could be the binding sites (epitopes) for disease causing antibodies in pemphigus (FIGURE 6). This explains the cross activity of disease causing antibodies against DSG3 and DSG4 from the results of Dr. Nguyen’s experiments in 1999 when he first found the evident of desmoglein 4 (FIGURE 9).
Note that DSG4 and DSG3 have similar sizes. Mature non-glycosylated DSG4 and DSG3 molecules have similar molecular weight of 130 kDa. Both proteins have the same glycosylated sites at their extracellular domain 1 and extracellular anchor domain.
DSG4 contains a cytoplasmic putative conserved signaling domain typical of classical cadherins (FIGURE 10). This consensus signaling region could function to induce clustering (sending signal into the cell to cluster proteins necessary to do cell function at the site such as desmosomes). It could bind to the signaling proteins protein such as p120ctn well as others that have been known to perform intracellular signal transduction. Without this characteristic, a DSG molecule unlikely could conduct any signal to cell. DSG3 does not have this feature.
Since the disease causing antibodies in pemphigus, at the cellular level, cause the disease by inducing signals to the cells that disrupt normal biological activities of skin cells. These signals cause internalization of DSG3 (and probably other cadherins such as desmocollin 3), retraction of cytoskeleton filaments causing unsupported desmosomes, or disadhesion between cells and blocking cell readhesion, etc…that ultimately lead to cells detach from one another, which is called acantholysis.
The signaling feature of DSG4 domain at the 3’ end may explain a part of the mechanism for the pathogenesis of the disease pemphigus. Hypothetically, the normal signals from the molecule control the clustering and organization of adhesion-related molecules and therefore control the dynamic of desmosomes during keratinocyte differentiation. Binding of disease causing antibodies to the molecule (as well as some other molecules, i.e., acetylcholine receptors) induce abnormal signals therefore disrupt this normal process and hence, participate to the pathomechanism of the disease.
Preliminary screening experiments showed that majority of pemphigus patients have antibodies against DSG4. Results from preliminary in vivo experiments showed that antibodies from pemphigus patients against DSG4 can cause pemphigus-like lesions in neonatal rat model (first developed by Dr. Nguyen) (unpublished data). This is a better model for pemphigus than the classic neonatal mouse model because genetically, with regard to desmogleins, similar to human, rat has 4 types of desmogleins, DSG1-DSG4; while mice has 2 additional desmoglein variants. Because of this important difference, the presence of 6 types of desmogleins in mice desmosomes, results of past neonatal mouse model for pemphigus might be inaccurate.
Some scientists might say the finding about the pemphigus pathogenicity of antibodies against DSG4 is just a cross reactivity with DSG3 and DSG1, or vice versa, pemphigus pathogenicity of antibodies antibodies against DSG1 and DSG3 is just a cross reactivity with DSG4. Either way, the important point is this discovery lets us understand better how the skin works and how the skin gets trouble.
The complicate pathogenesis of pemphigus at cellular and molecular levels gives scientists the opportunity to use it as a model to explore the nature of skin and see that skin is tough and that only one hit cannot knock it down. Using antibodies from pemphigus patients as the tool to discover novel human molecules, DSG4 is the third human molecules that Dr. Nguyen discovered before the end of human genome project (both proteins and genes- others are pemhaxin and alpha 9 nicotinic acetylcholine receptor, all together support his "Multiple Hit" theory.
The role of DSG4 in pemphigus: The history of the discovery of DSG4 and its scientific evidential facts clearly suggest the significant role of DSG4 in the pathogenesis of pemphigus. One could even think that the 130 kDa pemphigus antigen discovered by Dr. Stanley in 1982 is the same as the pemphigus 130 kDa antigen that Dr. Nguyen discovered in 2000, or the 130 kDa DSG4 that he discovered in 2002. However, immediately after Dr. Nguyen discovered DSG4 and its potential role in the pathogenesis of pemphigus, his study was abrupted by a sensitive and brutal publication in which authors including the one that reported the DSG4 variant 2 in November 2002 published an article to "define" the role of anti DSG4 autoantibodies in pemphigus in which antigenic epitopes in DSG4, DSG3, and DSG1 were used in their study. It concluded that PV autoantibodies to DSG4 had no role in the pathogenesis of the disease.
We all know that the nature defines our fates: born-grow up-age and die. We don't "define" the fate of nature. We don't create nature, we only discover what are already there. Similar to DSG3, DSG4 has many epitopes, pathogenic or non-pathogenic. If one simply wants to dispute the role of DSG4, just simply chose the non-pathogenic and nonsignificant one. Vice versa, if one seriously wants to test the role of DSG4, just simply chose the pathogenic and significant one, even if it is identical to that of DSG3 that was used to prove the pathogenic significance of DSG3. Thus if we put all other factors aside and seriously want to know nature, we should be more serious and truthful in our studies. But it would cost a lot of money and support. Dr. Nguyen discovered DSG4 on his own cost with no other support. May be some other scientists will continue the job in the future.
There was a study in 2007 in China that had strong evidence to support the significant role of DSG4 in the pathogensis of pemphigus vulgaris. It was not surprised that it could only be published on a Journal in China.
The "multiple hits" theory for the pathogenesis of pemphigus: With the preliminary findings from his study on pemphigus since 1996, in 1998 at a PLENARY session of the third International Congress of Investigative Dermatology in Cologne, Germany, Dr. Nguyen proposed his "multiple hits" hypothesis for the pathogenesis of pemphigus to explain the disease at the cellular and molecular levels. The theory postulates that acantholysis in PV results from simultaneous and cumulative effects of autoantibodies directed toward different keratinocyte self-antigens, including the “structural” antigens, such as desmosomal cadherins, and “signaling” antigens, such as cell surface receptors regulating function of the adhesion and cytoskeletal units, including acetylcholine receptors and desmogleins molecules structurally having the concensus signalling sequences. Desmoglein 4 acts as both "structural" and "signaling" antigen that adds more flavor to the theory (Nguyen et al, Journal of Biological Chemistry 2000; 275(38): 29466-76.)
Dr. Nguyen’s book chapter, CHAPTER 19 “Experimental Mouse Model of Pemphigus Vulgaris: Passive Transfer of Nondesmoglein 1 and 3 Antibodies“ in the book “Animal Models of Human Inflammatory Skin Diseases” (Editors: Dr. Lawrence S. Chan, Published by CRC in 2003) is the first and single publication that reports the first record of laboratory cloned DSG4, the GenBank AY168788. In this chapter, section D.1 “Identifying the novel pemphigus antigen DSG4“ describes how DSG4 was discovered.
Also in this book chapter, he proposed an hypothetical model how desmocollins and other desmogleins interact with desmoglein 4 to send the signals through Wnt/wingless pathway to control keratinocyte proliferation and differentiation, a process called "direct desmoglein-activated signaling". One possible explanation for the mechanism of PV is "unlock the door-block the door". The PV antibodies to DSG4 (and/or other signaling molecules) act as the key to unlock the adhesion between desmosomes (like unlock a door). Due to the dynamic of desmosomes, they could disadhese and adhese to one another again (like the door open then close again). PV antibodies to DSG3 act to block the readhesion of desmosomes (steric hindrance, like one step his foot to block the door, prevent it to close), resulting acantholysis in PV. This model could be used to support Dr. Nguyen's "multiple hit" theory.
The book chapter also points out the advantage and limitation of neonatal mouse model that researchers should be aware when reading old pemphigus studies.
Conclusion: Pemphigus vulgaris is a rare disease, but the nature of the disease and its pathogenesis has been an extremely interesting topic for many scientists. There is no simple answer for the disease but with the advance of science and technologies, there will be many exciting findings about the disease to see in the future.
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