**4. Sorting for regulated secretion in the parotid gland**

The parotid salivary gland provides an excellent model for the study of regulated secretion of proteins. This gland has evolved to secrete copious amounts of specific proteins into the saliva. It secretes salivary proteins including amylase, Parotid Secretory Protein (PSP), a family of acidic (aPRP) and basic (bPRP) Proline-Rich Proteins, and less abundant proteins such as histatin and statherin (Helmerhorst and Oppenheim 2007). Secreted PSP has antibacterial activity which contributes to protection of the oral cavity (Gorr et al. 2011). Human PSP (SPLUNC2, BPIFA2) has been shown to be expressed in saliva as several isoforms due to alternative splicing of the mRNA (Bingle et al. 2009; Bingle et al. 2011). Another abundant salivary protein, amylase, initiates digestion of starch, and also adheres to oral bacteria and enamel. PRPs contribute to secretion of other cargo proteins (Venkatesh and Gorr 2002; Venkatesh et al. 2007), are part of the acquired dental pellicle, and also bind bacteria. These three proteins are the most abundant luminal cargo proteins within the secretory granule. In addition, hundreds of other proteins are secreted into saliva by the parotid gland, and have been cataloged by proteomic approaches (Denny et al. 2008). As these proteins move through the trans-Golgi network, they are each presumably sorted into the correct pathway for secretion. We previously reviewed the pathways of sorting and secretion in the parotid gland (Gorr et al. 2005). Secretion in the parotid, as with other exocrine cells, includes the major regulated pathway, a minor regulated pathway, apical and basolateral constitutive secretory pathways, and a constitutive-like secretory pathway (Perez et al. 2010). Similarly, endocrine cells have both regulated secretory and constitutive secretory pathways, in addition to pathways within the cell (Kim et al. 2006; Park and Loh 2008). Of the major salivary proteins, PSP is an excellent marker for the regulated secretory pathway. Western blot analysis of rat serum demonstrates that some fraction of salivary amylase is normally present in serum, however, PSP was undetectable (Venkatesh et al. 2007). This indicates that under normal conditions PSP is tightly sorted into the apical regulated secretory pathway, whereas a portion of amylase enters a basolateral pathway *in vivo*.

Salivary glands are being studied for their potential to produce and secrete therapeutic proteins from transgenes introduced to patients (Perez et al. 2010). Towards this goal, it is important to understand the molecular mechanisms that control parotid sorting and secretion, in order to regulate whether the transgenic protein is secreted by an apical regulated (exocrine) or basolateral (endocrine) pathway (Perez et al. 2010). Progress has been made in defining molecular interactions that affect sorting in some cell types; however, many of these mechanisms do not appear to be present in parotid acinar cells. As described above, the pH of the secretory granules of neuroendocrine and endocrine cells decreases during maturation from about 6.2 at the TGN to 5.5 – 5.0 in the mature granule (Wu et al. 2001; Kim et al. 2006). This acidic environment is important for sorting in PC12 and AtT20 cells, and is essential for protein aggregation (reviewed in (Kim et al. 2006)). However, in the parotid gland, the pH of the acinar cell granule increases from about pH 6.2 at the TGN to 6.8 or higher after maturation (Arvan and Castle 1986). Furthermore, granule cargo proteins from parotid acinar cells (amylase and PSP) are unable to aggregate even in the presence of Ca++ and low pH, whereas pancreatic exocrine granule proteins (used as a control) aggregate in a fashion similar to endocrine cells (Venkatesh et al. 2004). This indicates that sorting of amylase and PSP in the parotid gland have at least some important differences from the mechanisms described for neuro/endocrine cells. Therefore, we have investigated

The parotid salivary gland provides an excellent model for the study of regulated secretion of proteins. This gland has evolved to secrete copious amounts of specific proteins into the saliva. It secretes salivary proteins including amylase, Parotid Secretory Protein (PSP), a family of acidic (aPRP) and basic (bPRP) Proline-Rich Proteins, and less abundant proteins such as histatin and statherin (Helmerhorst and Oppenheim 2007). Secreted PSP has antibacterial activity which contributes to protection of the oral cavity (Gorr et al. 2011). Human PSP (SPLUNC2, BPIFA2) has been shown to be expressed in saliva as several isoforms due to alternative splicing of the mRNA (Bingle et al. 2009; Bingle et al. 2011). Another abundant salivary protein, amylase, initiates digestion of starch, and also adheres to oral bacteria and enamel. PRPs contribute to secretion of other cargo proteins (Venkatesh and Gorr 2002; Venkatesh et al. 2007), are part of the acquired dental pellicle, and also bind bacteria. These three proteins are the most abundant luminal cargo proteins within the secretory granule. In addition, hundreds of other proteins are secreted into saliva by the parotid gland, and have been cataloged by proteomic approaches (Denny et al. 2008). As these proteins move through the trans-Golgi network, they are each presumably sorted into the correct pathway for secretion. We previously reviewed the pathways of sorting and secretion in the parotid gland (Gorr et al. 2005). Secretion in the parotid, as with other exocrine cells, includes the major regulated pathway, a minor regulated pathway, apical and basolateral constitutive secretory pathways, and a constitutive-like secretory pathway (Perez et al. 2010). Similarly, endocrine cells have both regulated secretory and constitutive secretory pathways, in addition to pathways within the cell (Kim et al. 2006; Park and Loh 2008). Of the major salivary proteins, PSP is an excellent marker for the regulated secretory pathway. Western blot analysis of rat serum demonstrates that some fraction of salivary amylase is normally present in serum, however, PSP was undetectable (Venkatesh et al. 2007). This indicates that under normal conditions PSP is tightly sorted into the apical regulated secretory pathway,

**4. Sorting for regulated secretion in the parotid gland** 

whereas a portion of amylase enters a basolateral pathway *in vivo*.

Salivary glands are being studied for their potential to produce and secrete therapeutic proteins from transgenes introduced to patients (Perez et al. 2010). Towards this goal, it is important to understand the molecular mechanisms that control parotid sorting and secretion, in order to regulate whether the transgenic protein is secreted by an apical regulated (exocrine) or basolateral (endocrine) pathway (Perez et al. 2010). Progress has been made in defining molecular interactions that affect sorting in some cell types; however, many of these mechanisms do not appear to be present in parotid acinar cells. As described above, the pH of the secretory granules of neuroendocrine and endocrine cells decreases during maturation from about 6.2 at the TGN to 5.5 – 5.0 in the mature granule (Wu et al. 2001; Kim et al. 2006). This acidic environment is important for sorting in PC12 and AtT20 cells, and is essential for protein aggregation (reviewed in (Kim et al. 2006)). However, in the parotid gland, the pH of the acinar cell granule increases from about pH 6.2 at the TGN to 6.8 or higher after maturation (Arvan and Castle 1986). Furthermore, granule cargo proteins from parotid acinar cells (amylase and PSP) are unable to aggregate even in the presence of Ca++ and low pH, whereas pancreatic exocrine granule proteins (used as a control) aggregate in a fashion similar to endocrine cells (Venkatesh et al. 2004). This indicates that sorting of amylase and PSP in the parotid gland have at least some important differences from the mechanisms described for neuro/endocrine cells. Therefore, we have investigated the molecular interactions of PSP in the secretory granule in an attempt to understand how sorting may be controlled (Venkatesh et al. 2011).

#### **4.1 Parotid secretory protein (PSP) binds to secretory granule membranes**

We analyzed rat parotid granule membranes by mass spectrometry with the goal of identifying integral membrane proteins that may be candidate sorting receptors in the parotid gland. Parotid gland homogenates were fractionated on sucrose gradients to isolate the secretory granules, which were osmotically lysed. Membranes were washed and further enriched by an additional sucrose gradient. Sucrose gradient-purified granule membranes were electrophoresed on polyacrylamide gels, and trypsinized peptides identified by MS/MS as described (Uriarte et al. 2008). Numerous integral or membranebound proteins were identified, including several involved in vesicle trafficking and cytoskeletal proteins, as expected. However, potential sorting receptors such as SgIII (Han et al. 2008) or carboxypeptidase E (Dhanvantari et al. 2002) were not identified in parotid membranes by this method. Nonetheless, one salivary cargo protein, PSP, was identified. Other abundant soluble cargo proteins such as amylase and acidic Proline-Rich Protein (PRP) were not detected. To confirm the binding of PSP to granule membranes, western blot analysis was done with independent preparations of purified and extensively washed granule membranes. This confirmed that PSP is selectively bound, whereas amylase and PRP are absent from purified membranes (Fig. 3) (Venkatesh et al. 2011). While this approach failed to identify candidate sorting receptor proteins, it did demonstrate that PSP is a good marker for interactions with the membrane. In other cell types, putative sorting proteins such as secretogranin III (Hosaka et al. 2004), carboxypeptidase E (Dhanvantari et al. 2002), PC1/3, and PC2 (Jutras et al. 2000) are also associated with isolated secretory granule membranes.

Fig. 3. Western blots of purified secretory granule membranes, probed with either antiamylase or anti-PSP. Lanes contain either intact granules (G), purified granule membranes (M), or soluble cargo protein lysate (L). Equal proportions (0.5%) of each fraction was analyzed. The star indicates PSP in the purified membrane fraction. Mw: molecular size standards.

Given the existing models for sorting receptors, we tested whether PSP was bound to a sorting receptor protein in the secretory granule membrane. Numerous experiments were done attempting to crosslink PSP to a membrane protein; however, PSP never crosslinked

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo 49

series of each of the seven different phosphorylated forms of PtdInsPs. We found that native PSP binds 3- to 5-fold more to PtdIns(3,4)P2 compared to PtdIns(4,5)P2 or PtdIns(3,4,5)P3, and 10-fold greater than PtdIns(3,5)P2 or PtdIns(4)P (Fig. 5). PSP does not bind PtdIns(3)P, PtdIns(5)P, or PtdIns. Half-maximal binding of PSP was with approximately 35 pmoles PtdIns(3,4)P2. Parallel blots failed to detect any bound amylase or acidic PRP, both of which are abundant in the granule lysates. This high degree of specificity indicates that PSP binds the head group of PtdInsPs, analogous to known PtdInsP-binding proteins (Di Paolo and De Camilli 2006). For example, PSP is more selective than the PH-domain protein DAPP1 which

binds PtdIns(3,4)P2 or PtdIns(3,4,5)P3 with similar avidity (Dowler et al. 2000).

Fig. 5. Binding of PSP to phosphatidylinositol phosphates. PtdInsP array membranes spotted with serially diluted lipids were used to define the binding of native PSP. Membranes were blocked and probed with anti-PSP as described (Venkatesh et al. 2011).

parotid granule proteins.

The experiments described above use a secretory granule lysate as the source of PSP. This leaves open the possibility that PSP binds indirectly to PtdInsP2. Therefore, PSP was expressed *in vitro* in rabbit reticulocyte lysates, and also was expressed in bacteria. Chloramphenicol acetyltransferase (CAT) was used as a negative control since CAT and rPSP are similar in size and also have similar acidic pI values. Rat PSP, and human PSP (human Splunc2, BPIFA2), each with a V5 tag, were translated *in vitro* and bound selectively to PtdIns(3,4)P2 and PtdIns(4,5)P2 demonstrating that this activity is conserved between species. CAT-V5 was unable to bind any of the lipids. Similarly, both human and rat PSP proteins were expressed in bacteria as glutathione-S-transferase (GST) fusion proteins having V5 tags. Again, GST-rPSP-V5 and GST-hPSP-V5 each bound strongly to PtdIns(3,4)P2 and did not bind PtdIns, whereas GST-V5 had no binding activity (Venkatesh et al. 2011). Bacterially expressed and GST-affinity purified rPSP-V5 also preferentially binds PtdIns(3,4)P2 (Fig. 6). The binding of *in vitro* synthesized, and bacterially expressed, rPSP and human PSP to PtdIns(3,4)P2 demonstrates that the interaction is independent of other

Pleckstrin homology domain proteins bind phosphoinositides with a moderate to high affinity (Vicinanza et al. 2008). We used bacterially expressed rPSP-V5 to determine the affinity of PSP for PtdIns(3,4)P2. Bacterially expressed affinity-purified rPSP-V5 was incubated at 0.1 to 3.5 μg/ml with membranes spotted with 50 pmoles of PtdIns(3,4)P2. Bound protein was detected with anti-V5 antibody, and the intensity of the signal used to calculate the amount of free and bound protein from a PSP-V5 standard curve, as described

into a specific membrane-dependent higher molecular weight band. Taking the opposite approach, we extensively digested parotid granule membranes with either trypsin or pronase to destroy all membrane-associated proteins, and subsequently found that exogenous PSP still binds quite effectively. These results indicated that PSP does not require a protein receptor for binding to the membrane. In contrast, exogenous amylase did not bind the trypsinized membranes, and was present in the unbound fraction only, emphasizing the specificity of the binding of PSP.
