Created by: Sunna Bashir

            Human gastric lipase(PBD ID: 1HLG), abbreviated HGL, is a globular protein that is secreted by the cells located in the stomach. It can handle the acidic environment of the stomach, and help with fat digestion (1). HGL is able to break down 10-30% of ingested triacylglycerols in the stomach. The hydrolysis of triacylglycerols can continue in the duodenum with the help of gastric and pancreatic lipases (2). Pancreatic lipases require bile acid or colipase for ideal enzymatic activity; however, HGL does not (3). HGL is fundamental for newborns, because it can penetrate the fat in the milk and help in the digestion of the fat globules. Newborns depend on their mothers for milk and need it for nourishment. If HGL is not present then there can be deficiencies present in the baby’s health. Babies up to 4 weeks old have not fully developed their pancreatic lipase, so HGL plays an important role in breaking down 50% of the ingested dietary fat (1). 

            The key function of HGL is its ability to act on an emulsified substrate. HGL can bind to a lipid droplets surface. This lipid droplet will be made up of triacylglycerols and free cholesterol. The break down of the lipid begins with the formation of free fatty acids, diacylglycerols, and a little monoacylglycerol. These degraded parts with go either to the surface or core of the droplet. There will be more monoacylglycerols and free fatty acids at the surface, causing the expansion of the surface of the lipid droplet. The pressure at the surface will increase causing the free fatty acids on the surface to rearrange into peripheral particles, and will trap HGL. The trapped HGL will not have access to the triacylglycerol, resulting in lipolysis inhibition. This will only last around 60 minutes because in its trapped form HGL can transfer from the hydrolyzed droplet to the surface of a new one (3).

            The ability of HGL to bind to lipids and break them down can be explained by its structure. The closed structure of HGL was obtained by X-ray diffraction with resolution of 3.00 Angstroms. HGL is a polypeptide that contains 371 residues in length. It is composed of alpha helices (41%), beta sheets (14%), and random coils. HGL contains two subunits. Subunits A and B are independent molecules arranged head-to-tail, and are related by a two-fold axis. These subunits have similar orientations, and their lids, that are covering the active site, are facing each other (1). HGL is a globular protein that has a core domain that belongs to the alpha/beta hydrolase-fold family, and it also contains a cap domain (1). HGL has a molecular weight of 42,361.18 Daltons, and isoelectric point of 6.50 (4). 

            There are four N-glycosylation sites in HGL (1). There location is at Asn-15, Asn-80, Asn-252, and Asn-308.  The N-glycosylation sites are associated with the sugar Asn at all four sites. N-Acetyl-D-Glucosamine (NAG) residues, the associated ligand with HGL, come in to maintain this alteration. NAG residues were introduced at Asn-80 and Asn-252. Asn-15, Asn-80, and Asn-252 are situated one the side of the molecule. On the other hand, Asn-308 is located on the other side of the protein. When looking at the structure, Asn-80 resides in the core of HGL, and Asn-252 is within the cap domain. These two N-glycosylation sites in HGL are close to each other, improving the relationship between the cap and core (1).  

            HGL has three important cysteine resides: Cys-244, Cys-227, and Cys-236. Cys-244 is a free residue, while Cys-227 and Cys-236 are involved in the disulfide bridge within the structure of HGL. Cys-244 is located near Ser-153 and His-353 (which are part of the catalytic triad). This cysteine residue is buried within the structure and allows for the binding of the mercury acetate derivative (1). It has been found that a cysteine residue can react stoichiometrically with loss of enzymatic activity in presence of some cysteine reagents. These findings suggest that this free residue cannot react in the catalytic mechanism for HGL. However, it was observed that an inhibition occurred with sulfhydryl reagents at this active site (1). 

            Three resides are present within HGL that make up the catalytic triad and they are: Ser-153, His-353, and Asp-324(1). Ser-153 is nucleophilic and has a ε conformation. This nucleophilic residue is buried under 30 residues. This is a lid belonging to the cap domain. Since the cap covers the nucleophilic serine, the fat droplets cannot access it all the time. The lid would have to move for the substrate to have contact with Ser-153. His-353 and Asp-324 are the other two residues in the catalytic triad that function together at the center of the active site. Overall, the catalytic triad does not have any more special features (1). 

            The oxyanion hole stabilizes the oxyanion transition state by hydrogen bonds with two main chain nitrogen’s. The hydrogen bond occurs between the first NH group of Gln-154 and the second NH group of Leu-67. It was found that the Leu-67 side chain could interact with the phosphonate inhibitors alkyl group (1). The C11-phosphonate inhibitor is placed in an active site to set the hydrophobic substrate-binding site (1). 

            The particular roles of the lid and cap domains during the catalytic step have not yet been explained clearly. There was a study done that investigates what the role of the cap and lid was. In the study, mutants of HGL were produced that lacked the cap and lid. It was found that their activity had decreased in comparison to the non-mutant HGL (5). This indicated that the cap and lid are important factors during the catalytic reaction. This study clarified the importance of the cap and lid and showed that they are involved in the binding of the fat droplets to the protein (5).  

            Overall, the structure of HGL is composed of three elements: the cap, the lid, and the core domains. The cap (residues 184-308) and lid (residues 210-267) are made up of helices. The core domain (residues 1-183, and residues 309-379) is made up of a central beta-sheet and six helices. The Ser-153 hides by the lid and cap domain in its closed form (5). When the lid is removed from HGL, the cap domain forms hydrophobic residues around the active site of the nucleophilic serine (Ser-153). This can act as a binding site, allowing fat droplets to attach (1, 5). 

            The closed form of HGL has been observed, and it was the first structure to be explained in the mammalian acid lipase family (1, 6). The open structure of Dog gastric lipase (PBD ID: 1K8Q) in Canis lupus, abbreviated DGL, was looked at to understand the functional significance of the cap and lid domains. DGL breaks down fats like triacylglycerol in the acidic stomach of a dog. The open structure of DGL was obtained by X-ray diffraction with resolution of 2.7 Angstroms (6). It was shown in complex with the undecyl-butyl (C11Y4) phosphonate inhibitor (2, 6). DGL has a structural similarity of 85.7% with HGL (6). DGL also has a similar primary and tertiary structure as shown by PSI-BLAST (E=0.00) and DALI (Z=54.3), respectively (7, 8). PSI-BLAST is a program used to find proteins that have the same primary structure close to the protein query. It assigns proteins an E value that shows the similarities to the query. If the value is less than 0.5 it indicates similarity. The E value was 0.00, which is less than 0.5 indicating that there is similarity to the primary structure. The Dali server looks at the tertiary structure of proteins and compares them. It calculates the differences in intramolecular distances. The Dali Server calculates the Z-score, which indicates if the proteins have similar folds if the value is greater than 2. The Z-score was 54.3, which is greater than 2 indicating a similarity in the tertiary structure. 

            The open DGL structure contains the catalytic triad, with the nucleophilic serine (Ser-153) buried within the structure, and also has the oxyanion hole depicted in HGL (1, 6). There are also differences between HGL and DGL. HGL has 2 subunits while DGL has 1 subunit (1, 6). HGL has 1 associated ligand (N-Acetyl-D-Glucosamine (NAG)) while DGL has 3 associated ligands (B-Octylglucoside (BOG), Undecyl-Phosphinic Acid Butyl Ester (C11), and N-Acetyl-D-Glucosamine (NAG)). Lastly, DGL binds to the C11Y4 phosphonate inhibitor that allows the cap domain, the lid, to move over and uncover the catalytic crevice. The C11Y4 inhibitor will go into the crevice and β-octyl glucoside (a ligand associated with DGL and not HGL) will fill up the crevice. With this occurrence, there is a possible binding site for fat molecules present (6). The movement of the lid is not simple, there are two events that occur during its opening. There is conformation reorganization, and a lot of movement. The closed and open forms of gastric lipase are mostly helical, but they have different numbers of helices. The closed lid has three helices while the open form has two helices. The opening of the lid occurs because of the rotation of the helices (6). Other than the lid, there are few differences observed between the open and closed forms. Overall, it can be assumed that the lid and cap help HGL selectively bind to fats, and allow the degradation of the fats. HGL can work in the acidic environment of the stomach to break down molecules like triacylglycerols, so that digestion in humans can occur correctly. The structure of HGL helps with its functional abilities, allowing it to attach to and break down fats.