Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta

Abstract

Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species.

Keywords: Clip domain; Hemolymph protein; Insect immunity; Phylogenetic analysis; RNA-Seq.

Fig. 1. Domain organization of 42 multi-domain SPs and SPHs in M. sexta

Signal peptide and other structural elements were predicted as described in Section 2.2. There are 27 single clip SPs, 6 single clip SPHs and 8 dual clip SPs represented by PAP1, SPH1 and PAP2, respectively.

Fig. 2. Phylogenetic relationships of the 125 nondigestive M. sexta SPs and SPHs

The amino acid sequences of the entire proteins were aligned for constructing the neighbor-joining tree as described in Section 2.2. On the basis of an arbitrary bootstrap value cutoff (>350 out of 1000 trials), arrowheads are placed at nineteen nodes closest to the tree center. These twigs are clockwisely numbered branches 1 through 19. Clip-domain SPs/SPHs in the CLIPA (★), CLIPB (○), CLIPC (▲) and CLIPD (♦) subfamilies are marked with the symbols, whose numbers correspond to the clip domain counts. The expression profiles (A, B, C, D, E and F, see Fig. S1) are indicated next to their names.

Fig. 3. Phylogenetic relationships of the catalytic/protease-like (A) and clip (B) domains of the 42 M. sexta CLIPs

Sequence alignments and neighbor-joining trees were constructed as described in Section 2.3. For proteins with multiple clip domains, these domains are numbered 1, 2, 3… Bootstrap values of greater than 200 in 1000 trials are marked at the corresponding nodes. Vertical bars and names (A:3, B:2, C:1a, D1:1b and D2:1c) indicate the CLIP (A to D2) and clip domain (1a to 3) groups.

Fig. 4. Molecular modeling of the clip domains in the 35 SPs and 7 SPHs of M. sexta

The 54 clip domains were modeled and compared as described in Section 2.4. (Sub)groups of the models and their representatives are shown in A through H and I through P, respectively. Panel A: the 1^st clip domain (group-2) in the CLIPB proteases GP33, HP12, HP15, HP23, HP24, HP26, PAP2, PAP3, and SPH42; Panel B: the 2^nd clip domain (group-2) in the same eight CLIPB proteases; Panel C: the clip domains (group-2) in the CLIPB proteases GP6, HP5, HP8 and PAP1; Panel D: the group-1a clip domains in the CLIPC proteases HP2, HP6, HP13, HP18a, HP18b, HP21, HP22, HP28, SP30, SP33 and SP144; Panel E: the group-1b clip domains in CLIPD1 proteases HP1a, HP1b, HP17a, HP17b, SP52 and SP60; Panel F: the group-1c clip domains in the CLIPD2 proteases SP131, SP132, SP140, SP141, SP142 and SP143; Panel G: the group-3 clip domains of SPH1a, SPH1b, SPH2, SPH4 and SPH101 (CLIPAs); Panel H: the five group-3 clip domains in SPH53 (CLIPA); Panels I through P: clip domains with the lowest RMSD in each (sub)group are HP24 clip domain-1 (I), HP24 clip domain-2 (J), PAP1 (K), HP13 (L), HP17a (M), SP132 (N), SPH1a (O) and SPH53-3 (P). The six Cys residues predicted to form three disulfide bonds (Cys1-Cys5, Cys2-Cy4, and Cys3-Cys6) are colored yellow, red, and magenta, respectively.

Fig. 5. Transcript profiles of the nondigestive SPs/SPHs in the 52 M. sexta tissue samples

Log₂(FPKM+1) values for the SP-related mRNAs are shown in the gradient heat map from blue (0) to red (≥10). The values of 0~0.49, 0.50~1.49, 1.50~2.49 … 8.50~9.49, 9.50~10.49 10.50~11.49, 11.50~12.49 and 12.50~13.49 are labeled as 0, 1, 2 … 9, A, B, C and D, respectively. Cluster analysis has revealed six distinct patterns. A (♦), B (□), C (△), D (▲), E (+), and F (●). The 52 cDNA libraries (1 through 52) represent the following tissues and stages: heads [1. 2^nd (instar) L (larvae), d1 (day 1); 2. 3^rd L, d1; 3. 4^th L, d0.5; 4. 4^th L, late; 5. 5^th L, d0.5; 6. 5^th L, d2; 7. 5^th L, pre-W (pre-wandering); 8. P (pupae), late; 9. A (adults), d1; 10. A, d3; 11. A, d7], fat body (12. 4^th L, late; 13. 5^th L, d1; 14. 5^th L, pre-W; 15. 5^th L, W; 16. P, d1–3; 17. P, d15–18; 18. A, d1–3; 19. A, d7–9), whole animals [20. E (embryos), 3h; 21. E, late; 22. 1^st L; 23. 2^nd L; 24. 3^rd L), midgut (25. 2^nd L; 26. 3^rd L; 27. 4^th L, 12h; 28. 4^th L, late; 29. 5^th L, 1–3h; 30. 5^th L, 24h; 31. 5^th L, pre-W; 32–33. 5^th L, W; 34. P, d1; 35. P, d15–18; 36. A, d3–5; 37. 4^th L, 0h), MT (38. 5^th L, pre-W; 39. A, d1; 40. A, d3), muscle (41. 4^th L, late; 42–43. 5^th L, 12h; 44–45. 5^th L, pre-W; 46–47. 5^th L, W), testes (48. P, d3; 49. P, d15–18; 50. A, d1–3), and ovaries (51. P, d15–18; 52. A, d1).