Silks are produced by a wide variety of arthropods including spiders and larvae of hundreds of insect species. Few silks are well-characterized, but emerging evidence suggests that differences in biochemical and biophysical properties are attributable to constituents that vary considerably among species(1) . In fact, silk proteins from silkworms(2, 3, 4) , spiders(5) , and aquatic larva of the midge, Chironomus tentans(6, 7) , are remarkably different.

The chironomid salivary gland is an exceptional system for the study of silk protein synthesis and assembly from gene to finished product(6, 7) . The polytene chromosomes are well-characterized and contain Balbiani rings (BRs), ()sites of intensive transcription of silk protein-encoding genes(6) . BR mRNAs that encode the 1000-kDa silk proteins are visible during transcription, packaging, transport, and translation(8, 9, 10, 11, 12) .

The nucleotide sequences for 13 major C. tentans silk proteins are known(6, 7) . These range in size from 12 to >1000 kDa, and their primary structures are highly unorthodox. Most consist of 50-150 tandem repeats of unusual amino acid sequences with a Pro- or Cys-containing motif, or both. Some of the silk proteins are glycosylated (13, 14) or phosphorylated(15, 16) , and all are secreted and stored in the lumen of the salivary gland. Proteins isolated from the lumen exist as soluble complexes, capable of disassembly and reassembly in vitro(17) . However, in vivo, the lumenal contents are pumped on demand through a long, thin salivary duct and exit the animal's mouth as an insoluble silk fiber. Nothing is known about the mechanism by which this phase transition occurs.

The ``special lobe'' of Chironomus salivary glands is an enigma. This lobe is present in many, but not all, species and contains four secretory cells, lying adjacent to the salivary duct(18) . The special lobe is distinct from the remainder of the gland: (i) its polytene chromosomes have one additional BR(18) , (ii) it contains one additional, special lobe-specific, silk protein(19, 20) ; (iii) its cells and secretion contain Beermann's secretory granules(18, 21) ; (iv) the lobe histochemically stains differentially for glycoprotein (22) . These observations led to the hypothesis that the special lobe-specific BR contains a gene encoding a glycosylated silk protein that is secreted in Beermann's granules and is responsible for the lobe's differential staining. Furthermore, the proximity of this lobe to the salivary duct suggests that these granules somehow contribute to silk protein assembly into fibers.

We report here the first step in testing this hypothesis in Chironomus thummi. A special lobe-specific cDNA was cloned that hybridizes in situ to special lobe-specific BRa. This cDNA encodes a protein with numerous sites for potential glycosylation. Antibody raised against a cDNA-encoded fusion protein reacts with ssp160, the only known special lobe-specific silk protein in C. thummi salivary glands(20) , and lectin binding demonstrates that ssp160 contains both N- and O-linked carbohydrate.

EXPERIMENTAL PROCEDURES

Salivary Gland Dissection

()

RNA Isolation and Blotting

cDNA Library Construction and Screening

cDNA Amplification and Labeling

()

In Situ Hybridization

Subcloning and Sequencing

Fusion Protein Expression and Purification

Antibody Production and Purification

Affinity-purified antibodies were obtained by chromatography (37) using 500 µg of gel-purified fusion protein coupled to Affi-Gel 10 (Bio-Rad). To remove antibodies against the ompT leader and co-purified E. coli proteins, affinity-purified antibody (14 µg of protein in 1 ml of 1% non-fat dry milk, 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.02% NaN) was adsorbed for 4 h at 20 °C with 275 µg of protein from a control lysate of isopropyl-1-thio--D-galactopyranoside-induced BL21(DE3)pLysS cells containing pET12b. The antibody was then diluted and used for Western blotting(38) . For competition studies, lysate from BL21(DE3)pLysS-containing pET12b with the cDNA insert (i.e. with fusion protein) was used for adsorption.

Western Blotting

Lectin Binding

To examine protein binding to lectins, special lobe proteins were extracted as described above (16) and dialyzed against TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) keeping the protein concentration less than 25 µg/ml to avoid precipitation. Dialysate was equilibrated with an equal volume of either GNA-Sepharose in TBS or PNA-Sepharose (EY Laboratories) in TBS plus 1 mM each MgCl, CaCl, and MnCl, for 2 h at room temperature with constant mixing. Supernatants containing unbound protein were removed, and the resin was rinsed thoroughly. To release bound protein, resins were then incubated for 1 h in 2 volumes of binding buffer containing 1 M mannose (GNA-Sepharose) (45) or 0.2 M lactose (PNA-Sepharose)(46) . The supernatant was removed, dialyzed against water, and proteins were precipitated with acetone. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted.

RESULTS

Cloning a Special Lobe-specific cDNA

Figure 1: Comparison of special and main lobe cDNA. P-labeled, double-stranded cDNA made from special (S) and main (M) lobe poly(A) RNA was electrophoresed on a 1% agarose gel and detected by autoradiography. An abundant 2.5-kb cDNA (arrowhead) was present only in the special lobe sample.

A radiolabeled special lobe-specific cDNA probe was made by subtractive hybridization against an excess of main lobe poly(A) RNA. To check probe specificity, we first hybridized the probe to a Northern blot containing special and main lobe RNA. Hybridization was seen only to a 2.5-kb special lobe RNA (Fig. 2A). This confirmed the lobe specificity of the probe and demonstrated that only one size class of special lobe-specific transcripts was detected.

Figure 2: Northern blot analyses of the special lobe-specific probe and cDNA clone. Five µg of special lobe (1) or main lobe (2) RNA were separated in parallel to RNA markers (numbers on left) by electrophoresis on denaturing 0.7% agarose gels, electroblotted to nylon membranes, and hybridized with the following radioactive probes. A, main lobe-subtracted, special lobe-specific cDNA; B, a 2.5-kb PCR probe from 160.1 cDNA; C, a 475-bp PCR probe for sp220 mRNA. A and B are different blots; however, C was made by rehybridization of B after radioactivity in the first probe was allowed to decay.

To determine the proportion of special lobe-specific transcripts in the entire gland, we screened two C. thummi whole gland cDNA libraries with the special lobe-specific subtracted probe. 1.7% of 1.2 10 clones in a primary gt23 library and 1.8% of 1.8 10 clones in an amplified ZAP library were hybridized. However, when this subtracted probe was used to screen the 2.5-kb size fraction gt22 library, 92% of the insert-containing clones hybridized. The first four clones selected had identical restriction enzyme cleavage patterns, suggesting that they are representative of cDNA molecules taken from the agarose gel. One, 160.1, was chosen for further characterization.

To determine the size of the RNA from which this cDNA was derived, primers complementary to flanking vector sequences were used to amplify and radiolabel the cloned 2.5-kb cDNA by PCR. Northern blots of special and main lobe-derived RNA showed specific hybridization only to a 2.5-kb RNA from special lobes (Fig. 2B). Subsequent rehybridization of this blot with a probe for 5.5-kb sp220 mRNA verified that lack of hybridization to main lobe transcripts was not due to RNA degradation (Fig. 2C). We conclude that the cloned cDNA is a near full-length copy of a 2.5-kb special lobe-specific RNA.

Localizing the Gene

Figure 3: Hybridization in situ of a digoxigenin-labeled 2.5-kb PCR probe from 160.1 to squashed preparations of C. thummi salivary gland polytene chromosomes. The probe was detected with fluorescein-conjugated anti-digoxigenin antibody. Phase contrast (left) and fluorescence (right) images of the same fields showing chromosome IV from C. thummi thummi (top) and C. thummi piger (bottom). The fluorescent band corresponds to the region A2b, the locus of BRa. Arrow, region A2b; arrowhead, BRb; N, nucleolus. Bar = 10 µm for all figures.

Determining the Primary Sequence

Figure 4: Nucleotide and deduced amino acid sequences of the special lobe-specific cDNA. Potential sites for N-linked glycosylation (underlined) are present. Thr, Ser, Ser, Thr, Ser, Thr, and Ser as well as a stretch of 21/25 threonine residues (663-689), located near the carboxyl end of the protein, are potential sites for O-glycosylation. Inverted triangles (nucleotides 1085 and 1373) indicate AluI sites bordering the fragment inserted into an expression vector for fusion protein production.

Analysis of the regions containing the NX(S/T) motifs reveals that region I is comprised of six almost-perfect tandem copies of the hexameric repeat TSSNST (Fig. 5). Region II has very similar hexamers; variations, including a 2-residue deletion, appear limited to the first three residues. Region III is separated from the others by the central basic core and is most divergent; however, seven 6-11-residue segments can be aligned by a conserved NXT motif. These patterns suggest that internal sequence duplication has occurred during evolution, preserving the NXT motifs.

Figure 5: Amino acid sequence of repeats in regions containing sites for potential N-linked glycosylation. Nineteen out of twenty-eight potential sites for N-linked glycosylation (NX(S/T) where XP) are located in these three regions.

While there is no definitive consensus for sites involved in O-linked glycosylation, residues that are so modified tend to be located +1 or -3 residues from a proline (e.g. P(S/T) or (S/T)XXP) or located near other serines, threonines, or alanines(43) . Based on these criteria, Thr, Ser, Ser, Thr, Ser, Thr, and Ser are possible candidates for O-glycosylation along with the numerous serines and threonines which neighbor each other.

Identifying the Encoded Protein

Figure 6: Western blots of salivary gland proteins reacted with affinity-purified anti-fusion protein antibody. Protein from special (1) and main (2) lobes was denatured, reduced, carboxymethylated, and separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were incubated with affinity-purified, anti-fusion protein antibody and binding was detected with an alkaline phosphatase-linked secondary antibody. A, total lobe (cells and secretion); color developed for 15 min. B, identical with A except that the affinity-purified antibody was preabsorbed with fusion protein; color developed for 40 min. C, purified secretion; color developed for 15 min.

Examining Carbohydrate Moieties

Figure 7: Lectin binding to special and main lobe proteins. Proteins extracted from special (1) and main (2) lobes of salivary glands were separated by electrophoresis and transferred to nitrocellulose. Membranes were stained with AuroDye (A) or reacted with digoxigenin-conjugated lectins: ConA (B), GNA (C), PNA (D). Bound lectins were detected with anti-digoxigenin antibody coupled to alkaline phosphatase. Arrowheads point to a 160-kDa protein present only in special lobe samples.

Figure 8: Western blots of lectin-purified salivary gland proteins reacted with affinity-purified anti-fusion protein antibody. Protein from special lobes was bound to, and released from, Sepharose-linked GNA (A) or PNA (B). The starting extract (1), supernatant remaining after depletion with lectin-Sepharose (2), and the supernatant-containing proteins released by either 1 M mannose (A) or 0.2 M lactose (B) from the lectin-Sepharose (3) were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Arrowheads point to the 160-kDa special lobe-specific protein.

DISCUSSION

Chironomid silk proteins have been studied most extensively in C. tentans. The molecular biological data base for the major proteins is complete(6) , and studies are underway to reveal how they fold and assemble into fibers(17, 48) . However, C. tentans evidently lacks a characteristic special lobe-specific BR and protein (18, 49) . The silk protein data base for species that have a special lobe (C. pallidivittatus(50) and C. thummi(51, 52) ) is small but growing(40) , but comparable data for special lobe proteins is hard to come by. Salivary glands in these species are smaller (1 mm in length), and yields of special lobe poly(A) RNA (2 ng/lobe) and silk proteins (<100 ng/lobe) are limited. This project required the manual dissection of over 4000 salivary glands. Nonetheless, a special lobe-specific cDNA has been acquired, and the encoded protein has been identified.

We conclude that 160.1 cDNA encodes the major, if not only, special lobe-specific silk protein in C. thummi salivary glands. Its mRNA is lobe-specific (Fig. 2) and cDNA most abundant (Fig. 1). Its gene resides in lobe-specific BRa (Fig. 3). The encoded immunoreactive protein is absent from main lobes and found in special lobes and their secretion (Fig. 6). These characteristics fit criteria defining ssp160(20) ; however, there is a discrepancy between the calculated (77 kDa) and apparent (160 kDa) molecular mass of these proteins ( Fig. 4and Fig. 6). The presence of 77-kDa dimers is possible but unlikely since, for Western blotting, protein extraction included denaturation in 6 M guanidine HCl, reduction of disulfide bonds, and covalent modification of cysteinyl sulfhydryl groups. An alternative explanation is that decreased electrophoretic mobility is due to protein glycosylation. For example, N- and O-linked sugars comprise 50-70% of the mass of some glycoproteins(53) . ssp160 can incorporate label from [H]glucosamine(20) , and the amino acid sequence inferred from the cDNA (Fig. 4) contains numerous sites for potential glycosylation. Lectin binding coupled with Western blotting experiments showed that the 160-kDa special lobe-specific immunoreactive protein contains both O- and N-linked sugars ( Fig. 7and Fig. 8). These results indicate that the cDNA-encoded and immunoreactive proteins are the same. To estimate how much glycosylation contributes to the mobility of this protein, we attempted enzymatic removal of sugars followed by immunoblotting with anti-fusion protein antibody to detect the resulting protein core. Despite successful removal of sugars from purified control proteins and lack of detectable proteolysis, repeated attempts to deglycosylate silk proteins were inconclusive; lectin binding was abolished, but we failed to detect an immunoreactive band. Since the bacterially expressed fusion protein could not have contained carbohydrate epitopes, the evident loss of immunoreactivity could not be due to loss of sugars per se. A more plausible explanation is that detection was hampered by the heterogeneous distribution of partially deglycosylated products. Thus, we conclude that 160.1 cDNA does, in fact, encode ssp160.

The primary structure of ssp160 is novel. A survey (54) of data bases revealed no overall similarities but some related regions. For example, ssp160's putative hydrophobic leader peptide (MNIKVILVCALVAIFFA) resembles that of Drosophila cuticle protein precursors (e.g. MFKILLCALVALVAA) (55) . Sequences similar to ssp160's threonine-rich region are found in Drosophila laminin A chain(56) , glutactin(57) , and human ankyrin(58) . Whether or not the threonines in these proteins are glycosylated is unknown. ssp160 is unusual even among chironomid silk proteins. These proteins are composed nearly entirely of blocks of tandemly repeated sequences(6) . ssp160's repeats are few, comprising only a small portion of the protein. Since ssp160 lacks the Pro- and Cys-containing motifs characteristic of most other silk proteins, its gene would likely be of independent evolutionary descent, including limited internal sequence duplication of regions preserving the NXT motifs (Fig. 5). sp240/420 of C. tentans also lacks the Pro- and Cys-containing motifs, is rich in serine and threonine, and is N-glycosylated(14) ; however, its numerous tandem repeats lack any resemblance to ssp160.

Chironomid salivary glands are dedicated to producing large amounts of a small ensemble of silk proteins. This is reflected in the cDNA banding pattern seen for both special and main lobes; several distinct cDNAs are common to both lobes, and their sizes coincide with mRNAs for small and midsize silk proteins. The amount of ssp160 cDNA (the 2.5-kb special lobe-specific band in Fig. 1) is remarkable, prompting expectations of proportionally high levels of ssp160; however, Coomassie-stained gels ()and AuroDye-stained blots (Fig. 7) indicate that neither ssp160, nor any other special lobe-specific protein, accumulates to such levels. This contrasts with other silk proteins whose steady-state level of mRNA and protein coincide(59, 60, 61) . These observations suggest that either translational control of ssp160 synthesis or its half-life in the gland differs dramatically from that of other silk proteins.

The role of the special lobe and ssp160 is uncertain. There are no reported differences in either tube-building behavior, structure of silken feeding/pupation tubes, or properties of silk in species that do and do not have special lobes. The molecular probes and antibodies acquired in this study will enable us to examine Beermann's granules for the presence of ssp160 and begin a phylogenetic investigation of the evolution and expression of ssp160-encoding genes that may lead to elucidation of the function of the special lobe.

FOOTNOTES

*

This work was supported in part by National Science Foundation Grant MCB-9204837 (to S. T. C.) and Graduate Fellowship GER-9253887 (to R. T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U24265[GenBank].

§

To whom correspondence should be addressed: Dept. of Biochemistry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505. Tel.: 601-984-1518; Fax: 601-984-1501; stcase{at}fiona.umsmed.edu.

()

The abbreviations used are: BR, Balbiani ring; spX, silk protein with an apparent molecular mass of X; ssp160, special silk protein 160; PCR, polymerase chain reaction; ConA, concanavalin A; GNA, Galanthus nivalis agglutinin; PNA, peanut agglutinin; kb, kilobase(s); bp, base pair(s).

()

For simplicity, we refer to the nonspecial lobe portion of the gland as the ``main lobe'' because those cells all appear similar to each other. However, anatomical distinctions between different regions of the gland have been made(7) .

()

S. T. Case, C. Cox, W. C. Bell, R. T. Hoffman, J. Martin, and R. Hamilton, manuscript in preparation.

()

R. T. Hoffman and S. T. Case, unpublished data.

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