Comment and Reply: Nematode EXP
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Comment on: 
Plant degradation: a nematode expansin acting on plants

Qin et al. (Nature 427: 30, 2004) report that a nematode protein with sequence similarity to expansin has wall extension activity characteristic of expansin. They conclude that this protein, named Gr-EXP1, has "the structural and functional characteristics that define the expansin superfamily". 

But is this really an expansin?

Structural characteristics: Gr-EXP1 has two domains. The first domain is resenbles a carbohydrate binding module family II (CBM-II). This domain is not found in plant expansins. The second domain has significant sequence similarity to plant expansin domain I. This domain is part of a large superfamily of domains that are found in a wide variety of proteins - most notably glycoside hydrolase family-45 (GH45) which includes a number of potent endoglucanases. Members of GH45 have been found in a variety of organisms, especially fungi, but also a bacterium, a protist living in the hindgut of termites, and a mollusk.  This domain gets around! It is identified as 50685 in SCOP (ver. 1.63) and is part of many different kinds of proteins, not just expansins and GH45 enzymes, but also barwin/hevein-like/PR4 proteins (plant defense proteins), a plant signaling protein known as PNP, and a variety of genes of unknown function in many phyla. The sequence of Gr-EXP1 is so distant from that of plant expansins, that if we accept it as an expansin, then all the proteins within this domain superfamily (SCOP 50685) might logically be classified as expansins. But many of them have distinctive functions.

Functional characteristics:  Expansins (a) induce rapid extension of heat-inactivated cell walls, (b) accelerate stress relaxation of cell walls and (c) lack significant wall hydrolytic activity. Quin et al. presented evidence that Gr-EXP1 induces wall extension, although only crude mixtures containing Gr-EXP1 were tested. They did not present data pertaining to (b) and (c). So, at this point it is unclear whether Gr-EXP1 has true expansin activity. Endoglucanases can induce wall extension, so it is a possibility that Gr-EXP1 caused wall extension through this mode of action.  It is also possible that the wall extension activity was not due to the expansin-related domain, but to the CBM-II domain.

Thus, I conclude that Gr-EXP1 does not have characteristic expansin structure (this has recently been defined more explicitly - after the publication of Quin et al; see Nomenclature). Whether it has all the functional characteristics of expansin is unclear. It might just be a very interesting form of GH45.

Daniel Cosgrove,
Penn State University
email: dcosgrove@psu.edu

19 February 2004 (revised)

Response from the authors

Writing a paper for Nature implies that one has to deal with very stringent space limitations. As a consequence we could present only a part of the information we current have about an expansin from the potato cyst nematode Globodera rostochiensis. Here I would like to comment on some of your concerns:

> (DC) But is this really an expansin?
> Structural characteristics:
> 1. Gr-EXP1 has two domains. The first domain is resembles a carbohydrate binding module family II (CBM-II). This domain is not found in plant expansins.

As described in the Cosgrove nomenclature ("defined more explicitly - after the publication of Qin et al"), expansins domain II is a grass pollen group-2 allergen-like domain (SCOP 1.63 49590) - GPG2. This is in fact also a carbohydrate-binding module being a cellulose-binding domain [Henrissat, Teeri and Warren (1998) FEBS Lett, 425,352-354 and Barre and Rougé (2002) BBRC 296,1346-1351].
[DC comments: the Henrissat et al. publication says nothing about this domain and the second article relies entirely on unsupported speculation by me (!) regarding the function of this domain. To my knowledge there are no published data showing that this domain has carbohydrate-binding activity and it is not included in the list of 39 carbohydrate-binding modules in Henrissat's CAZy database]  Moreover sequence to 3D structure alignment shows that the closest template of Gr-EXP1-D1 is a xylan-binding domain from Cellulomonas fimi (1xbd), with 17% sequence identity, 25% sequence homology and an e-value of 0.005, the second template is a cellulose binding domain [found exo-1,4-beta-D-glycanase from Cellulomonas fimi (1exg)] with 16% sequence identity, 23% sequence homology). Both belong to Cellulose-binding domain family II (according to SCOP classification). This suggests that Gr-EXP1-D1 may – just like plant expansin domain II - be involved in plant cell wall matrix polysaccharide adhesion, performing at molecular level a similar function with GPG2.

> (DC - continuation “Structural characteristics”) > The second domain has significant sequence similarity to plant expansin domain I. > This domain is part of a large superfamily of domains that are found in a wide > variety of proteins - most notably glycoside hydrolase family-45 (GH45) which > includes a number of potent endoglucanases. Members of GH45 have been found in > a variety of organisms, especially fungi, but also a bacterium, a protist living in the > hindgut of termites, and a mollusk. This domain gets around! It is identified as > 50685 in SCOP (ver. 1.63) and is part of many different kinds of proteins, not just > expansins and GH45 enzymes, but also barwin/hevein-like/PR4 proteins (plant > defense proteins), a plant signaling protein known as PNP, and a variety of genes > of unknown function in many phyla. The sequence of Gr-EXP1 is so distant from > that of plant expansins, that if we accept it as an expansin, then all the proteins > within this domain superfamily (SCOP 50685) might logically be classified as > expansins. But many of them have distinctive functions.

Indeed, SCOP families are rather large as they define only the overall topology of the protein and do not refer to details (such as: the length of the sequence, the number and length of secondary structure elements, the accessibility profile, the S-S bond pattern or the presence of various local sequence motifs involved in function). However the statement that "if we accept Gr-EXP1 as an expansin, then all the proteins within this domain superfamily (SCOP 50685) might logically be classified as expansins" is not justified. By taking in account the details mentioned below and performing sequence to 3D structure alignments one can easily spot with high confidence the real structural homologues. In our case, even by eye inspection one could easily find the best templates within the SCOP family (see attachment). More elaborate techniques, indicate that the closest template of Gr-EXP1-D2 is indeed the pollen allergen phl p1 from Phleum pratense, N-terminal domain (PDB code: 1n10, plant expansin domain 1) with ~25% identity and an e-value of 3.18e-06 (corresponding to more than 95% fold recognition confidence). With ~14% identity and an e-value of only 1.57e-01 (corresponding to less 80% fold recognition confidence), the second relative (the Barwin protein) comes way below 1n10 as possible structural homologue. In addition Barwin lectin is rather a single domain protein and similar sequences could not be found as part of multi-domain proteins.

On short, therefore, Gr-EXP1 has 2 domains with similar functionality to the 2 domains of plant expansins: -1) a CBM and -2) a plant expansin domain I. In contrast to the plant expansins however, the domains are swapped and the CBM could possibly recognise xylan rather than cellulose (additional experiments needed). Based on these structural arguments we think that Gr-EXP and Pl-EXPs are similar, and that the structural definition "Only two-domain proteins will be designated as members of the expansin superfamily. The name "expansin" should not given to proteins that are homologous to only one of the expansin domains, i.e. the GH45-like domain I (SCOP 1.63 50685) and the grass pollen group-2 allergen-like domain II (SCOP 1.63 49590)." is rather arbitrary, eliminating domains that could perform similar (but slightly different) functions at molecular level - as for example targeting different parts of the complex plant wall†.
[DC comments: “A rose by any other name would smell as sweet.” The definition of expansins based on sequence may not be completely congruent with that based on sequence. Because it is not feasible to assay the activity of every protein, we are left with the less-than-perfect method of classifying based on sequence. With multi-domain proteins, the classification problem is severely aggravated. Crystal structure data convincingly show that expansin domain 2 is a structural homologue of GH45 enzymes, a subset of SCOP 50685. Our tests show that GH45 enzymes lack significant expansin activity, so clearly not all proteins with GH45 domains have expansin activity. There is no natural structural demarcation between the expansin domain 2 and the catalytic domain of GH45 enzymes. When one wants to classify proteins containing a domain as widespread and divergent as the GH45-like domain, one must draw lines somewhere, and sometimes well-intentioned people will disagree on where the line should be drawn.]

(† This hypothesis is included in Cosgrove et al. (1995) PNAS, 92, 9245-, p. 9249)

> (DC) Functional characteristics: >it is also possible that the cell wall extension activity was not due to the expansin-related domain, >but to the CBM-II domain”.
Sure, carbohydrate-binding modules may also induce disruptions of the non-covalent interactions in cellulose microfibrils. Therefore, it could indeed be argued that the cellulose-binding domain in Gr-Exp1 is responsible for the cell wall-loosening activity observed in the extensometer assays. However, transgenic tobacco expressing the CBM of Gr-Exp1 alone showed no cell wall-loosening activity (not significantly different from the empty vector control plants). Primary data will be included in a follow-up paper.

> (DC) Expansin ... (c) lack significant wall hydrolytic activity
We checked recombinant Gr-EXP1, and - while this protein showed a potent cell wall expansion activity - no endoglucanase activity could be detected in standard assays.

Hans Helder & Andrei Petrescu

- Hans Helder (Lab. Nematology, Wageningen University, The Netherlands), e-mail: Hans.Helder@wur.nl - Andrei Petrescu (Institute of Biochemistry of the Romanian Academy, Bucharest, Rumania), e-mail: ap@biochim.ro

03 March 2004 (revised)

 

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