(Received for publication, December 20, 1995; and in revised form, March 8, 1996)
From the
Preservation of cell viability and function in the hyperosmolar environment of the renal medulla is a complex process that requires selective gene expression. We have identified a new member of the heat shock protein (hsp) 70 superfamily that is up-regulated in renal inner medullary collecting duct cells (mIMCD3 cells) during exposure to hyperosmotic NaCl stress. Known as osmotic stress protein 94, or Osp94, this 2935-base pair cDNA encodes an 838-amino acid protein that shows greatest homology to the recently discovered hsp110/SSE gene subfamily. Like the hsps, Osp94 has a putative amino-terminal ATP-binding domain and a putative carboxyl-terminal peptide-binding domain. The in vitro translated Osp94 product migrated as a 105-110-kDa protein on SDS-polyacrylamide gel electrophoresis. In mIMCD3 cells, Osp94 mRNA expression was greatly up-regulated by hyperosmotic NaCl or heat stress. In mouse kidney, Osp94 mRNA expression paralleled the known corticomedullary osmolality gradient showing highest expression in the inner medulla. Moreover, inner medullary Osp94 expression was increased during water restriction when osmolality is known to increase. Thus, Osp94 is a new member of the hsp110/SSE stress protein subfamily and likely acts as a molecular chaperone.
The renal inner medulla accumulates high concentrations of NaCl
and urea to enable the kidney to generate a concentrated urine. This
harsh hyperosmolar environment is known to adversely affect cellular
functions such as protein biosynthesis(1, 2) .
Cellular adaptation to hyperosmolar stress is a complex process
requiring enhanced expression of selected
genes(2, 3, 4) . Renal epithelial cells
exposed to hyperosmolar NaCl accumulate organic osmolytes through
enhanced expression of genes encoding aldose reductase for synthesis of
sorbitol as well as transporters for uptake of betaine, inositol, and
taurine (3, 4) . Hyperosmolar NaCl is also known to
augment expression of stress proteins such as hsp70, ()grp94, and
-crystallin in renal and nonrenal
cells(5, 6, 7, 8) . Moreover, hsp70
mRNA expression was modulated by the organic osmolyte betaine during
states of hypertonic stress with the implication that organic osmolytes
act to stabilize cellular biochemical processes during such
stresses(6) . Although substantial work has elucidated the
roles of organic osmolyte-related genes during hyperosmotic stress,
there is little known about the roles of heat shock proteins in this
setting.
As a class, stress proteins are considered to function as molecular chaperones in which they transiently bind to other proteins and facilitate folding or other biochemical processes(9, 10, 11) . Many families of stress proteins have been identified such as hsp70, hsp90, and hsp60, and numerous studies have examined their roles in stress adaptation and normal cellular function(12) . Recently, Lee-Yoon et al.(13) cloned a new stress protein, hsp110, and found through homology analysis that it is a member of a major subfamily of large hsp70-like proteins that was previously unrecognized. Termed the hsp110/SSE subfamily, it includes the structurally similar proteins hsp110 and hsp70RY (14) that both possess structural features of molecular chaperones. Hsp110 was found to be ubiquitously expressed and greatly induced by heat stress(13) , whereas the cellular role of hsp70RY remains largely unknown. The hsp110/SSE gene subfamily was shown to also contain several more distantly related genes including the sea urchin sperm receptor(15) , an orphan ORF from Caenorhabidits elegans(16, 17) , and two genes known as SSE1 and SSE2 from yeast(18, 19) . Thus, the hsp110/SSE gene subfamily is conserved evolutionarily and appears to include stress-responsive molecular chaperones.
We recently began to identify differentially expressed mRNAs in renal epithelial cells exposed to hyperosmolar NaCl. Among the differentially expressed mRNAs we identified was an hsp110 homolog which we have named Osmotic Stress Protein 94 kDa or Osp94. The present study describes the cloning and sequencing of Osp94 as well as an analysis of its expression in renal epithelial tissue. In sum, Osp94 is a new member of the hsp110/SSE subfamily and is induced by hyperosmotic stress and heat shock.
Reamplified cDNA fragments were separated on a 2% agarose gel,
isolated, blunt-end ligated into pBluescript SK, and
cloned. Plasmid DNA was isolated and the cDNA insert was sequenced at
the Howard Hughes Biopolymers Research Facility at Harvard Medical
School.
Figure 1:
A. Side-by-side comparison of
differential mRNA expression in mIMCD3 cells exposed to isotonic (Control) or hyperosmotic NaCl (+NaCl) medium.
The arrow indicates the differentially expressed 423-bp PCR
fragment of Osp94 that was isolated and sequenced. B, Northern
analysis of Osp94 expression in mIMCD3 cells exposed to isosmolar (Control) or hyperosmolar NaCl (+100 mM) for 24
h. Each lane contained 2 µg of poly(A) RNA. Not
shown, equal loading of the lanes was confirmed by probing with
GAPDH.
To
obtain a full-length cDNA, a directional cDNA library was prepared and
screened using the PCR fragment as a probe. A full-length 2938-bp cDNA,
named Osp94, ()was isolated and found to contain a 2514-bp
open reading frame and a 38-bp poly(A) tail. (
)Contained
within the Osp94 cDNA sequence is the PCR product extending from
nucleotide 894 to 1289, excluding the primers.
The 2.9-kb Osp94 cDNA appears to correspond to the 2.9-kb transcript detected on Northern analysis (Fig. 1B). To determine whether the larger 4.1-kb transcript represented an alternative polyadenylation splice variant containing additional 3`-UTR sequence we performed 3` RACE. As shown in Fig. 2, 3` RACE generated two products, one matching the predicted 380-bp size that would result from the 2.9-kb transcript and a larger 1600-bp product as would be predicted for a 4.1-kb mRNA. No 3` RACE product was detected that would appear to derive from the larger, less abundant 11.2-kb transcript seen in Fig. 1B.
Figure 2: 3` RACE analysis of Osp94 mRNA transcripts. RT-PCR was performed using total RNA from mIMCD3 cells treated with NaCl for 24 h, and the PCR products were separated in 2% agarose and visualized by ethidium bromide staining. Two products (A and B) with approximate sizes of 380 and 1600 bp were detected corresponding to the sizes predicted for the 2.9- and 4.1-kb Osp94 transcripts. Lanes are as follows: L, DNA ladder; 1, without cDNA; 2, without reverse transcriptase; and 3, cDNA + reverse transcriptase.
In vitro translation of Osp94 (Fig. 3) generated one prominent protein product with an apparent molecular mass of 105-110 kDa, somewhat greater than the mass of 94,278 Da calculated from the deduced protein. Also detected were several smaller protein products. In vitro translation in the presence of canine pancreatic microsomes had no major effect on the size or abundance of the 105-110-kDa protein product, although there appeared to be small changes in the smaller, minor products. The significance of the minor products is unknown, although they may represent inefficient translation start sites or, less likely, cleavage sites. Overall, these data suggest that the major protein product of Osp94 has a molecular mass of 105-110 kDa on SDS-PAGE.
Figure 3:
In vitro translation of Osp94
analyzed by SDS-PAGE. Lane 1 (-cDNA) shows a
negative control using water instead of the cDNA. Lanes 2 and 3 show S-labeled protein products when Osp94 cDNA
was translated in the absence or presence of pancreatic microsomes (micr).
Based on the cDNA sequence the deduced Osp94 protein (Fig. 4) contains 838 amino acids. The deduced protein contains putative myristylation sites and many consensus phosphorylation sites for protein kinase C, casein kinase 2, and tyrosine kinase. Hydrophobicity analysis using either the Kyte-Doolittle or Eisenberg algorithm indicated there are no transmembrane domains suggesting it is localized intracellularly. Whereas the subcellular localization of Osp94 remains to be defined, analysis of Osp94 using PSORT (22) indicated it may possess signal sequences that target the protein to mitochondria and peroxisomes but not nucleus though the degree of certainty (0.3-0.47) appeared to be relatively low. ARSGGI (amino acids 18-23) may represent a potential mitochondrial matrix signal and ARK (amino acids 123-125) may be a potential peroxisomal signal sequence, although this remains speculative. There is no evidence of Osp94 containing a signal peptide sequence. The deduced protein contains 15 cysteines localized predominantly in the amino half of the protein. Moreover, the deduced protein is highly charged, particularly the carboxyl terminus.
Figure 4: Deduced amino acid sequence of Osp94 (accession no. U23921) and its alignment with two homologous proteins, hsp110 (13) (Z47807) and hsp70RY (14) (L12723). Shaded areas indicate amino acid identities among the three proteins. Overlined amino acids indicate the putative five motifs of the ATP binding domain and correspond, in order, to the segments known as phosphate 1, connect 1, phosphate 2, adenosine, and connect 2 (23) .
Like many other members of the hsp family of proteins, Osp94 has two structurally distinct domains: an amino-terminal putative ATP-binding domain and a carboxyl-terminal putative peptide-binding domain. The overlined amino acids in Fig. 4indicate the five putative motifs of the ATP-binding domain that form a three-dimensional pocket for ATP binding and a putative interdomain hinge region that couples the ATP binding domain to the substrate binding domain comparable to that found in a large class of ATPase molecules including the hsp70s, actin, and hexokinase(23) . The carboxyl-terminal half of Osp94 contains no distinct consensus structural motifs. However, sequence homology to known peptide binding chaperone molecules suggests it functions as a peptide/protein binding domain.
Figure 5:
Tree of homology of several mammalian
hsp70 and hsp110 family members based on the unweighted pair group
method with arithmetic mean algorithm. Deduced amino acid sequences of
mouse Osp94 (GenBank accession no. U23921), hamster hsp11
(Z47807), human hsp70RY (L12723), mouse hsp70 (M35021), mouse hsc70
(M19141), and mouse BiP (P20029; Swiss-Prot) were compared. The lengths
of the horizontal lines are proportional to the estimated
genetic distances. The numbers indicate the average percentage
identity among different proteins beyond the branch
point.
An extensive homology analysis
showed Osp94 is most similar to hsp110 (65% identical) (13) and
hsp70RY (62% identical)(14) . Osp94 also has lower but
significant homology (30%) to the smaller hsp70 class of chaperones
including the inducible (hsp70) and constitutive forms (hsc70) as well
as the endoplasmic reticulum chaperone known as BiP or
grp78(25) . Consistent with this observation, analysis of
PROSITE protein sequence motifs showed Osp94 contains two of the three
hsp70 protein family signatures. In particular signature 2
(FIDMGHSAYQVSVC) and signature 3 (IEIVGGATRIPAVKE) were present in the
amino half of the deduced protein. As for the hsp70 signature 1 motif,
Osp94 contained the first 4 amino acids (IDLG) but failed to conserve
the terminal half of the motif (TTXS). Finally, as noted by
Lee-Yoon et al.(13) for hsp110 but not shown in Fig. 4, the extracellular domain of the sea urchin sperm
receptor(15) , hypothetical 86.9-kDa protein from C.
elegans, YLA4CAEEL(16, 17) ,
and two ORFs from yeast known as SSE1 and SSE2(18, 19) are also homologous to Osp94.
Figure 6: Time course of Osp94 mRNA expression during hyperosmotic stress. Total RNA was isolated from mIMCD3 cells exposed to isosomolar(-) or hyperosmolar NaCl (+) for 0.5 to 72 h. Upper figure shows Osp94 mRNA expression was transiently increased with maximal expression observed at 12-24 h. Lower figure shows GAPDH mRNA level was relatively unchanged. The larger 11.2-kb Osp94 transcript observed in Fig. 1B is not shown.
Since Osp94 is structurally related to the hsp70 superfamily it was of interest to determine whether Osp94 was also inducible by heat shock. As shown in Fig. 7, heat shock increased Osp94 expression in a manner similar to that seen with other heat stress inducible mRNAs. mRNA expression increased within 1 h of heat shock, was maximal at 3 h, and returned to approximately control levels at 24 h. Not shown, tunicamycin, a well known inducer of BiP expression, failed to elicit an increase in Osp94 expression. Thus, Osp94 is a heat shock-inducible gene akin to hsp70.
Figure 7: Time course of Osp94 mRNA expression during heat stress. RNA was isolated from mIMCD3 cells exposed to normal (37 °C) or elevated (42 °C) temperatures for 0.5 to 24 h. Northern blots containing 20 µg of total RNA were probed with a full-length Osp94 cDNA probe (upper figure) or a GAPDH cDNA probe. Upper figure shows Osp94 mRNA expression was greatly increased with maximal expression observed at 3 h. Lower figure shows GAPDH mRNA level was relatively unchanged.
Figure 8: Expression of Osp94 mRNA in kidneys from control and dehydrated mice. Control mice were provided ad libitum access to water, and dehydrated mice were water-restricted for 24 h to induce an increase in renal inner medullary osmolality. Total RNA was prepared from three regions of the kidney: cortex, outer medulla, and inner medulla. Northern analysis (10 µg of total RNA) shows increasing Osp94 expression from cortex to inner medulla. Dehydrated mice expressed elevated levels of Osp94 mRNA in inner medulla. GAPDH mRNA levels were relatively unchanged.
Osp94 is an hyperosmotic and heat stress-inducible member of the recently described hsp110/SSE gene subfamily(13) . The members of this gene subfamily have emerged as a relatively large and evolutionarily conserved group that includes structurally related genes from mammals, yeast, C. elegans, and sea urchin. With the exception of the sea urchin sperm receptor, the members of this subfamily have no known functions but their structural similarities to hsp70, hsc70, and BiP provide indirect evidence they function as molecular chaperones.
Osp94, like hsp110 and hsp70RY, possesses two
structurally distinct domains. The N-terminal half contains a putative
ATP-binding domain that is characteristic of other members of this gene
subfamily. Moreover, like hsp70, hsc70, and
BiP(27, 28) , the C-terminal portion of Osp94 contains
a putative peptide-binding domain. Within this region of the hsp70
family of molecules a variable, substrate recognition domain has been
characterized (10, 27, 29) whose tertiary
structure shows similarity to the variable peptide-binding domain of
the antigen presenting major histocompatibility complex class I
proteins(30) . Two portions of this C-terminal region within
Osp94 (Val-Gln
and
Lys
-Asp
) contain nonconserved amino
acid sequences that failed to show significant homology to any
proteins, including hsps, in the sequence data bases. The functional
significance of the C-terminal domain including identification of
potential peptide ligands which may be bound by Osp94 remains to be
elucidated and will likely reveal the functional role of Osp94 and
other members of the hsp110/SSE subfamily.
In vitro translation of Osp94 showed it migrated on SDS-PAGE with an apparent size of 105-110 kDa, somewhat greater than its calculated molecular mass of 94 kDa. This observation is consistent with the fact that hsp110 has a calculated molecular mass of 96,042 Da which is less than the 110-kDa size observed on Western analysis (13) .
Northern analysis showed three distinct Osp94 transcripts whose expression levels varied in synchrony. The cloned 2.9-kb cDNA corresponds to the 2.9-kb mRNA and 3` RACE suggested that the 4.1-kb transcript contains a larger 3`-UTR indicative of an alternative polyadenylation site. The significance of the minor 11.2-kb transcript remains to be elucidated, but it is reasonable to hypothesize that it is derived from the same gene and encodes the same protein but contains a larger 3`-UTR. At present, there is no evidence of alternative splicing within the coding region of Osp94.
Northern analysis delineated the response of Osp94 to hyperosmotic stress. In mIMCD3 cells, hyperosmotic NaCl transiently increased Osp94 expression. This response is similar to that observed previously for hsp70(5, 6) . Although the mechanism of induction is unknown, it is noteworthy that this level of hyperosmotic stress is known to suppress protein synthesis(1) , and disruption of protein synthesis is a known stimulus of hsp70 expression(9) . Moreover, the transient expression of Osp94 mRNA correlates with the known rate of accumulation of protein-stabilizing organic osmolytes in renal epithelial cells, suggesting that the stress proteins serve a cytoprotective role during the period of high intracellular salt stress that precedes organic osmolyte accumulation(2, 31) .
Heat shock also induced mRNA expression indicating Osp94 is a heat shock-inducible gene like hsp70. Similarly, hsp110 was shown to be induced by heat shock in CHO cells(13) , whereas the response of hsp70RY to heat stress is unknown. The failure of tunicamycin to induce Osp94 expression distinguishes it from BiP and other endoplasmic reticulum localized stress proteins suggesting Osp94 is not associated with endoplasmic reticulum-mediated functions. Hsp110 was found to be localized primarily in the cytoplasm and the periphery of nucleoli (13) . The subcellular localization of Osp94 is unknown at this time and will undoubtedly be an issue of significant interest in the future. Moreover, whether other stressors can induce expression of Osp94 and other members of the hsp110/SSE subfamily remains to be explored.
Analysis of renal expression of Osp94 provided direct evidence that this gene is responsive to osmotic stress in vivo. The renal urine concentrating mechanism requires the inner medulla to be hyperosmotic. In rodents, such as mouse, inner medullary osmolality is approximately 1000 mOsm under control conditions, and with water restriction osmolality rises severalfold over the course of 1-3 days (26) . This hyperosmotic inner medullary milieu is comprised in large part of a high concentration of NaCl. In control mice, Osp94 mRNA expression showed a cortical-medullary gradient profile that paralleled the known renal osmolality gradient. Moreover, with 24 h of water restriction, Osp94 expression was significantly elevated in renal inner medulla consistent with its activation by hyperosmotic stress.
In conclusion, Osp94 is a new member of the hsp110/SSE gene subfamily. Osp94 is inducible by hyperosmotic stress and heat shock. The physiological and biochemical functions of Osp94 are unknown but it, like other members of the larger hsp70 superfamily of genes, likely acts as a molecular chaperone.