Lysosomes, which may originate from pre-existing endolysosomes or autolysosomes (Mahapatra et al., 2021), are spherical bodies with amorphous of 50–500 nm diameter, single membrane, terminal degradation vesicular structures organelles, and are found in almost all eukaryotic cells (Debjyoti et al., 2014; Rondón-Barragán et al., 2017). Lysosomes are responsible for the degradation of cellular macromolecules (such as carbohydrates, proteins, lipids, and nucleic acids) into their constituent building blocks, which are generally obtained via phagocytosis or endocytosis, or autophagy (Alessandrini et al., 2017; Baba et al., 2020). Accumulating evidence indicated that lysosomes act as a metabolic signalling centre and play an essential role in numerous physiological progresses, such as, nutrient sensing (Perera and Zoncu, 2016; Lawrence and Zoncu, 2019), antigen presentation (Watts, 2012), disease development (Nishino et al., 2000; Ballabio and Gieselimann, 2009; Appelqvist et al., 2013; Alessandrini et al., 2017) and metabolic homeostasis (Schneede et al., 2011; Lim and Zoncu, 2016; Gu et al., 2021). In addition, lysosomes contain not only a number of soluble acid hydrolases, but also plenty of integral lysosomal membrane proteins (Wilke et al., 2012). Among these proteins, lysosome-associated membrane proteins (lamps) are the major ones, accounting for nearly half of the total lysosomal membrane proteins (Eskelinen, 2006; Rajapakshe et al., 2015; Lee et al., 2016). And these proteins are involved in a variety of complex tasks, including the acidification of the lysosomal lumen, lysosomal trafficking, autophagosome-lysosome membrane fusion, exocytosis, and transport of degraded products to the cytoplasm (Saftig and Klumperman, 2009; Huang et al., 2017).

The lamps gene family consists of five members, lamp1 (lgp120, CD107a) and lamp2 (lgp110, CD107b, LGP96), lamp3 (CD63, CD208, DC-LAMP, TSC403), lamp4 (CD68, macrosialin) and lamp5 (BAD-lamp) (Alessandrini et al., 2017). It has been reported that lamp1-deficient mice are fertile and viable (Andrejewski et al., 1999), but lamp2 deficiency in mammals, such as in the mice or humans, leads to postnatal mortality (Tanaka et al., 2000), Danon disease (Nadeau et al., 2008), fatal cardiomyopathy and myopathy (Tanaka et al., 2000; Stypmann et al., 2006) due to a massive accumulation of autophagic vacuoles in many tissues, including skeletal muscle, liver, spleen and kidney (Tanaka et al., 2000). Previous studies have shown the function of lamp3, lamp4 and lamp5 in the human immune response, for example, lamp3 is not only involved in recruited to Salmonella pathogens for intracellular proliferation (Lee et al., 2016), but overexpression lamp3 induced lysosomal membrane permeabilization and contributes to cell death in human salivary glands (Tanaka et al., 2022; Nakamura et al., 2023b). The lamp4 gene may have the capacity of negative regulatory functions in antigen presentation processing (Song et al., 2011). In addition, one study has shown that lamp5 is an essential regulator of inflammatory-signaling (Lee et al., 2014). Although different isoforms of lamp have been found, and they have different functions in mammals. Few studies have been conducted in teleosts, which urgently need to explore the function of these genes.

The lamp families are highly evolutionary conserved. Structurally speaking, all lamps belong to the type I transmembrane proteins and are composed of a large, highly glycosylated luminal ectodomain with multiple N- and O-glycosylation sites (Dominguez-Bautista et al., 2015), which protects them from the action of proteolytic degradation in lysosomes (Baba et al., 2020), and a transmembrane domain, followed by a short C-terminal cytoplasmic tail (Wilke et al., 2012; Terasawa et al., 2016). In addition, the C-terminal cytoplasmic tail has tyrosine-based sorting signal motifs (-G-Y-X-X-hydrophobic residue, X, an amino acid), consisting of 11 amino acid residues that function through adaptor proteins that are recognized to be involved in trafficking of membrane vesicle coating between the trans-Golgi network and the lysosomal membrane (Baba et al., 2020; Sakane and Akasaki, 2018).

To date, lamp gene families have been isolated and identified from several animal species, including mouse (lamp1 and lamp2) (Hughes and August, 1981), human ((lamp1 and lamp2) (Fukuda et al., 1988), lamp3 (Salaun et al., 2003), lamp4 (Holness and Simmons, 1993) and lamp5 (Defays et al., 2011)) and bird (lamp3) (Wu et al., 2010). In fish, however, the lamp genes have received far too little attention in the literature. Only four orthologs of mammalian lamp genes, including lamp1, lamp2, lamp3 and lamp4 have been characterized in zebrafish (Danio rerio) (Meireles et al., 2018; Iyer et al., 2022), lamp1 was identified in japanese flounder (Paralichthys olivaceus) (Rondón-Barragán et al., 2017), lamp3 was isolated from rainbow trout (Oncorhynchus mykiss) (Johansson et al., 2012) and lamp4 was cloned from bluntnose black bream (Megalobrama amblycephala) (Cui et al., 2022). And the above results showed that lamp family genes play a vital immune role in teleosts.

It is well known that the fish growth and basic physiological progress are influenced by extrinsic and intrinsic factors, such as, temperature, pH, salinity, dissolved oxygen, size, age, reproductive or nutritional status (Reinecke, 2010; Fan et al., 2019; Song et al., 2019; Alfonso et al., 2021; Zengin, 2021; Liu et al., 2023). In particular, starvation stress is considered to be one of the most important intrinsic factors that not only alters the immune function (Tran et al., 2018; Song et al., 2019; Liao et al., 2021), but also changes basic energy metabolism in teleosts (Dai, W. et al., 2018; Dar et al., 2019; Florescu Gune et al., 2019; Martin et al., 2010). For instance, in Sinibrama taeniatus, the innate immune parameters were increased, but the adaptive immune index was declined after short period of starvation (Shi et al., 2023). In addition, starvation changes the plasma glucose level and increased lysozyme levels in Anguilla anguilla (Caruso et al., 2010). Furthermore, a series of studies have found that nutrient deprivation stress also induceed the autophagy in teleosts (Yabu and Yamashita, 2008; Yabu et al., 2012). For examples, many scholars indicated that the autophagosome formation was observed in the skeletal muscle and intestine of juvenile Chinese Perch (Siniperca chuatsi) after a short-term starvation (Wu et al., 2020; Wu et al., 2022; Pan et al., 2022). Fan and others (2020) demonstrated that nutrient deprivation induced autophagosome formation in the zebrafish embryonic fibroblast cells. Meanwhile, plenty of autophagy-related genes were also induced (Wu et al., 2022). For instance, previous studies have shown that the transcript levels of autophagy-related genes (LC3B, gabarapl1, atg12l, atg4b) were significantly induced by 14days fasting or serum deprivation of rainbow trout myocytes (Seiliez et al., 2010). Similarly, Wu et al. found that the expression of autophagy-related genes (Atg4b/c/d, Becn1, bnip3, LC3a, LC3b, Gabarapl1, and Atp6v1d) was significantly increased in muscle after treatment with nutrient deprivation in Chinese perch (Wu et al., 2020; Wu et al., 2022). Other studies have shown that nutrient restriction greatly increased the expression of Atg genes (atg4, atg9, atg12, lc3, gabarap and becn1) in gill epithelial cells of O. mykiss (Walbaum) (Balmori-Cedeño et al., 2019). A recent study reported that starvation stimulates lamp1 expression in zebrafish embryonic fibroblast cells (Fan et al., 2020). However, further research and exploration are needed to study the lamp family genes under food-restricted conditions in teleosts.

Largemouth bass, Micropterus salmoides, belongs to family Perciformes, order Centrarchidae, and is one of the most important economic carnivorous fish species in China (Hu et al., 2022). It is quite popular with customers due to its lack of intermuscular bone, tasty flesh and high nutritional value (Zhu et al., 2022). However, like most of fish species, largemouth bass often suffered from food shortage and/or starvation throughout its life history due to the seasonal variations, reproduction, breeding migration and overwintering (Zou et al., 2023). To cope with the above serious problem, the autophagy-lysosomal pathway is usually initiated in fish (Belghit et al., 2014; Séité et al., 2018; Lu et al., 2019). However, few studies have focused on the effect of starvation on the lysosomal membrane proteins in largemouth bass.

In the present study, the full-length coding sequences of three lamp genes (lamp1, lamp2, lamp3), were isolated and characterized from the largemouth bass. Subsequently, the tissue distribution patterns of these lamp genes were measured to investigate their properties and functions. Meanwhile, we also determined their transcription in response to different nutritional status. Our results may provide new insights for a better understanding of the function of lamp genes in the responses of fish species to food deprivation and/or starvation.

In the current study, three of the lamp family genes (lamp1, lamp2 and lamp3) of largemouth bass (M. salmoides) were successfully identified and characterized. The full-length coding sequences of lamp1, lamp2 and lamp3 were 1,251, 1,221 and 771bp, and predicted to encode a protein of 416, 406 and 256 amino acids, respectively, which is consistent with previous findings, such as in japanese flounder (Bandyopadhyay et al., 2014; Rondón-Barragán et al., 2017), zebrafish (GenBank no. NM_001013533) and rainbow trout (Johansson et al., 2012), indicating that the family of lamp1-3 genes may be conserved in fish. The multiple sequence alignment revealed that LAMP1-3 proteins contain a typical feature of a type I transmembrane (a signal peptide, a long extra-cellular domain, a hydrophobic transmembrane domain and a short cytoplasmic domain), protein cysteine residues and proline/threonine region, that is highly conserved among vertebrates, showing that LAMP1-3 proteins of M. salmoides may have the same function in other species. In addition, a conserved tyrosine-based sorting signal motif (YXXØ sorting signal) located at the carbon terminal was also observed, which is capable of targeting the lysosome (Guarnieri et al., 1993). Interestingly, the N-end of the LAMP3 protein was missing in all teleosts, which is similar to a previous report carried out in the sequences of the trout, salmon and zebrafish (Johansson et al., 2012), suggesting that the functional conservation of lamp3 in fish.

To investigate the evolutionary history of lamp1-3 genes in vertebrates, a phylogenetic analysis was conducted based on the protein sequences, selected from these representative species. We observed that the relationship between M. salmoides and other teleosts and vertebrates in the constructed phylogenetic tree is in consistent with traditional systematics (Figure 4). Meanwhile, the phylogenetic analysis showed that the LAMP1, LAMP2 and LAMP3 were clustered together with the homologous counterparts from other fishes and vertebrates, and divided into three subclades, which is in line with a previous study carried out by Johansson (Johansson et al., 2012).

In the current study, mutil-copies lamp (1–3) genes in M. salmoides were located on different chromosomes and possessed variable numbers of exons and introns (Figure 5), which is likely to be due to gene duplication events (Morash et al., 2010). Interestingly, the number of exons and introns of lamp3 is much lower than that of lamp1 and/or lamp2, further suggesting that these lamp genes may have different physiological functions in fish species. This is consistent with many studies in mammals, for instance, previous studies found that the lamp2-deficiency is closely associated with Danon disease, which is characterized by developmental disability, cardiomyopathy and myopathy in mammals (Malicdan et al., 2008; Nishino, I et al., 2000). Sakane and Akasaki (2018) demonstrated that Lamp1 was involved in myoblasts differentiation. Meanwhile, lamp3 was found to be involved in the regulation of diseases associated with hepatic lipid metabolism disorders (Liao et al., 2018).

Gene synteny and gene structure results showed that conserved gene clusters, i.e., cul4a-lamp1-grtp1-adprhl1-dcun1d2-tmco3, clgaltlc1-mcts1-cul4b-lamp2-atp1b4-tmem255a-zbt33, lamp3-mccc1-dcun1d1-atp11b-sox2, which belonging to lamp1-3 (Figures 6A–C), respectively, were observed in almost representative vertebrate genomes, implying that the lamp1-3 genes may be exhibited a highly conserved synteny during evolution. However, some flanking genes of the M. salmoides lamp1-3 were arranged differently or lost compared with the other fishes or vertebrates (Figure 6), which might be the result of the gene deletion or gene rearrangement event (Hu et al., 2022), and showing that the largemouth bass has an independent evolutionary history.

Exploring the mRNA distribution patterns of lamp1-3 genes in different tissues would be beneficial to understand the role of these genes in largemouth bass. The tissue expression patterns of the three genes were analyzed by qRT-PCR. The results showed that the lamp1 gene was widely distributed in all tissues examined, and the highly transcript level of lamp1 was found in the brain (Figure 7A), which is in line with previous studies in Japanese flounder (Rondón-Barragán et al., 2017), indicating that the lamp1 gene may be involved in neural activity. For instance, Barrachina et al. (2006) revealed that the transcript level of lamp1 was increased in neurons and in glial cells surrounding senile plaques in patients with Alzheimer’s diseases. Furthermore, the high expression of lamp1 mRNA in the muscle suggested that it may also be involved in myogenesis, which was consistent with a study carried out by Sakane and Akasaki (2018), who demonstrated that knocked down of lamp1 decreased the expression levels of myogenic regulatory factors in C2C12 myotube formation. However, the function of lamp1 in other tissues and its expression pattern in fish is still unknown.

In vertebrates, lamp2 mRNA expression has been documented in several tissues/organs, such as neural crest derived ganglia, liver, pancreas and kidney in murine (Lichter-Konecki et al., 1999), heart and brain in bird (Hatem et al., 1995), and C2C12 myoblasts in mouse (Sakane and Akasaki, 2018). Similarly, lamp2 transcription was extensively expressed in all selected tissues in largemouth bass, and abundantly exhibited in heart, muscle and spleen, indicating that it might may play a critical role in these tissues. In human, accumulating evidences revealed that lamp2 deficiency induced hypertrophic cardiomyopathy and eventually led to the Danon disease (Zhai et al., 2023). In addition, previous studies showed that lamp2 was involved in the differentiation process of C2C12 myoblasts to myotube (Sakane and Akasaki, 2018). Whether the lamp2 has the same function in teleosts requires further investigation.

In contrast to the ubiquitous lamp1 and lamp2, lamp3 mRNA expression has been reported to be tissue and species specific. For instance, in mammals, previous studies have observed that higher expression of lamp3 in various cancer tissues (carcinomas) (Nagelkerke et al., 2011; Wang et al., 2017; Gui et al., 2018; Nakamura et al., 2023a). Salaun et al. (2003) have shown that mouse lamp3 mRNA is expressed almost exclusively in peripheral cells of the lung, but is not detected in other tissues. de Saint-Vis et al. (1998) have found that lamp3 expression levels are induced upon activation of human dendritic cells. Other studies have indicated that the chicken lamp3 is expressed in a wide range of tissues, including lung, thymus, spleen, bursa and ceacal tonsil (Wu et al., 2010). In the present study, we observed that Ms-lamp3 was constitutively distributed in all tissues examined, with the highest expression in spleen, and higher in liver and kidney tissues, which is consistent with the study by Johansson et al. (2012), which revealed that the lamp3 transcription was highest in spleen, kidney and liver of O. mykiss, and was induced by infection with viral and bacterial pathogens, referring that lamp3 may play a critical immune role in largemouth bass. In addition, the lamp3 was highly expressed in the heart, we speculated that it also has a pivotal role in this tissue, but its need further study.

Many studies have been clearly elaborated that the starvation or food-deprived often influenced the function of immunity in mammals (Han et al., 2021; Loft et al., 2022; Forslund, 2023; Janssen et al., 2023) and altered their energy metabolism (Browning et al., 2012; Soeters et al., 2012; Dote-Montero et al., 2022). Similarly, the aforementioned phenomena have also been observed in teleosts (Dai et al., 2018; Liao et al., 2021; Wang et al., 2021; Gou et al., 2023; Shi et al., 2023). Moreover, although some studies have shown that starvation alters the expression of the autophagy-related genes in fish (Balmori-Cedeño et al., 2019; Wu et al., 2020; Pan et al., 2022), there are still many autophagy-related gene from teleosts that have not been identified and characterized. In our study, we observed that the expression of lamp1 in the liver was significantly induced after starvation, and then returned to normal levels after refeeding (Figure 8A). This finding was consistent with previous studies in zebrafish (Fan et al., 2020), suggesting that the lamp1 gene may be involved in energy regulation via autophagy in largemouth bass. To validate that autophagy occurred after long-term starvation, the transcription level of two classical autophagosomal markers, Beclin1 and MAPLC3 (Yabu et al., 2012; Al-Shenawy, 2016), was detected in three tissues (liver, spleen and kidney) of largemouth bass in the present study. As expected, the mRNA levels of both of autophagosomal marker genes were significantly increased in the three tissues after the fasting treatment, and then remarkably decreased after the refeeding treatment (Figure 9). Interestingly, the lamp2 and lamp3 showed the same expression pattern trends as lamp1 in the liver tissue (Figures 8D, G). The liver is a multi-functional organ with roles in metabolism, nutrient storage, innate immunity and detoxification in vertebrates (Robinson et al., 2016; Causey et al., 2018). The high expression levels of lamp genes in the liver were induced by long-term food deprivation, which might facilitate the fish to maintain energy homeostasis during starvation. Collectively, we speculated that lamp1-3 genes might be involved in energy regulation in the liver via the autophagy pathway in largemouth bass and these results provide a novel insight into the mechanism of the adaptive starvation in this fish species.

To investigate whether or not the lamp1-3 genes are involved in the immune progression in largemouth bass, we determined their transcriptional patterns in response to starvation and refeeding schedules. In the present study, the expression of lamp1 in the largemouth bass kidney was remarkably decreased after starvation and refeeding compared to the control group. On the contrary, the transcript level of lamp2 and lamp3 in kidney was significantly increased after a long-term food deprivation, and then strikingly decreased to the same level or even lower than that of the control group after refeeding (Figures 8F, I), suggesting that the lamp2 and lamp3 but not the lamp1, play a priority role in the kidney tissues under the stress condition (long-term food deprivation) via autophagy. In addition, we found that there was no significant difference in the mRNA expression levels of both lamp1 and lamp3 in the spleen of largemouth bass between the control and fasted group (Figures 8B, H), but the transcript level of lamp2 was remarkably increased in the fasted treatment group and slightly decreased in the refed treatment group (Figure 8E), indicating that the lamp2 might play an important role in the spleen. The above expression patterns were similar to previous findings of IL-8 in kidney and IL-6 and IL-4 in the spleen piglets after dietary restriction treatment (Han et al., 2018). Kidney is an important haematopoietic tissue with the ability to sample and retain antigens (Bjørgen and Koppang, 2021) and plays a vital role in immunity. The spleen is considered to be a primordial secondary lymphoid organ, generating adaptive immune responses in almost all gnathostomes (Flajnik, 2018). Previous studies have been demonstrated that the autophagy-related genes were also induced in the kidney and spleen when fish were challenged with viruses (Kong et al., 2011; Zhang et al., 2022), bacteria (Qin et al., 2016) or stress conditions (Kumar et al., 2022; Kumar et al., 2023). It is therefore reasonable to speculating that the lamp2 and lamp3 genes may also have an important immunity function in largemouth bass, which needs to be further investigated.

In summary, the lamp1-3 gene family of largemouth bass (M. salmoides) has been identified and characterized. Multiple alignment of protein sequences, 3-D structural model prediction, gene synteny and phylogeny were performed, showing that the lamps genes are conserved across vertebrates. In addition, the distribution of these genes was analyzed by q-PCR, indicating that three genes are extensively detected but showed different patterns. Furthermore, the fasting and refeeding schedule experiments demonstrated that three lamp genes may have different functions in immune and energetic organisms in largemouth bass.